ORIGINAL RESEARCH Open Access Functioning of drug ...Results: It was found that heme-centered...

ORIGINAL RESEARCH Open Access

Functioning of drug-metabolizingmicrosomal cytochrome P450s: In silicoprobing of proteins suggests that the distalheme ‘active site’ pocket plays a relatively‘passive role’ in some enzyme-substrateinteractionsAvanthika Venkatachalam1, Abhinav Parashar2 and Kelath Murali Manoj1,2,3*

Abstract

Purpose: The currently held mechanistic understanding of microsomal cytochrome P450s (CYPs) seeks that diversedrug molecules bind within the deep-seated distal heme pocket and subsequently react at the heme centre. Toexplain a bevy of experimental observations and meta-analyses, we indulge a hypothesis that involves a “diffusibleradical mediated” mechanism. This new hypothesis posits that many substrates could also bind at alternate loci on/within the enzyme and be reacted without the pertinent moiety accessing a bonding proximity to the purportedcatalytic Fe-O enzyme intermediate.

Methods: Through blind and heme-distal pocket centered dockings of various substrates and non-substrates (drugmolecules of diverse sizes, classes, topographies etc.) of microsomal CYPs, we explored the possibility of access ofsubstrates via the distal channels, its binding energies, docking orientations, distance of reactive moieties (or moleculeper se) to/from the heme centre, etc. We investigated specific cases like- (a) large drug molecules as substrates,(b) classical marker drug substrates, (c) class of drugs as substrates (Sartans, Statins etc.), (d) substrate preferencesbetween related and unrelated CYPs, (e) man-made site-directed mutants’ and naturally occurring mutants’ reactivityand metabolic disposition, (f) drug-drug interactions, (g) overall affinities of drug substrate versus oxidized product, (h)meta-analysis of in silico versus experimental binding constants and reaction/residence times etc.

Results: It was found that heme-centered dockings of the substrate/modulator drug molecules with the available CYPcrystal structures gave poor docking geometries and distances from Fe-heme centre. In conjunction with several otherarguments, the findings discount the relevance of erstwhile hypothesis in many CYP systems. Consequently, the newlyproposed hypothesis is deemed a viable alternate, as it satisfies Occam’s razor.(Continued on next page)

* Correspondence: [email protected] at PSG Institute of Advanced Studies, Avinashi Road, Peelamedu,Coimbatore, Tamil Nadu 641004, India2Formerly at Hemoproteins Lab, School of Bio Sciences and Technology, VITUniversity, Vellore, Tamil Nadu, India632014Full list of author information is available at the end of the article

© 2016 Venkatachalam et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made.

Venkatachalam et al. In Silico Pharmacology (2016) 4:2 DOI 10.1186/s40203-016-0016-7

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(Continued from previous page)

Conclusions: The new proposal affords expanded scope for explaining the mechanism, kinetics and overallphenomenology of CYP mediated drug metabolism. It is now understood that the heme-iron and the hydrophobicdistal pocket of CYPs serve primarily to stabilize the reactive intermediate (diffusible radical) and the surface or crypts ofthe apoprotein bind to the xenobiotic substrate (and in some cases, the heme distal pocket could also serve the latterfunction). Thus, CYPs enhance reaction rates and selectivity/specificity via a hitherto unrecognized modality.

Keywords: Cytochrome P450, Heme-enzymes, Mechanism, Substrate binding

BackgroundThe cytochrome P450 (CYP) family of enzymes possessesthe heme-thiolate functionality and they mediate the phaseI metabolism of a vast majority of drugs and xenobiotics inmost animals, including man (Testa 1995). Many of thesereactions are known to be regiospecific and some of themare even enantioselective (Martinez and Stewart 2000).Since the hydroxylation of several non-activated substratesare not noted with the more commonly found heme-histidylate proteins, CYPs’ catalytic mechanism called for adefined and selective process, which the protein’s "activesite" could afford. Therefore, the fundamental step in thecatalytic mechanism invoked the formation of high poten-tial intermediate(s) centered at the heme-thiolate moiety,involving an iron - oxygen species (Ortiz de Montellano2015; Meunier et al. 2004; Denisov et al. 2005; Volz et al.2002). This proposal was along the lines of a two-electrondeficient catalytic species identified as Compound Iobserved in heme peroxidases (Raven and Dunford 2015).Thereafter, the substrate’s interaction with this enzymeactive intermediate is understood to occur by an ‘oxygen-rebound’ mechanism (Groves 1985). As per the prevailingunderstanding, the formation of this catalytic CYP inter-mediate solicits a highly fastidious, multi-step, orderedprocess involving termolecular complexations of CYPs withcytochrome P450 reductase (CPR), cytochrome b5 (Cyt. b5),substrate, molecular oxygen etc. (Guengerich and Isin2008). Such a catalytic cycle requires that the diversesubstrates (herein considered as the final oxygen atomacceptor) bind to a given distal heme pocket of a CYP atthe very first step (and stay bound till the very end), toinduce a redox potential change of the heme-iron for theoverall cycle (for protein-protein electron transfer, substratehydroxylation and superoxide/peroxide/water production)to be feasible (Guengerich and Isin 2008). Therefore, theprevailing CYP catalytic mechanism obligatorily espouses ahigh-affinity binding and positioning of the diverse sub-strates at a favorable locus within the distal heme pocket(also known as the “active site”). When the substrate getsconverted to the product, the latter is supposed to lose af-finity for the enzyme and hence, it detaches and diffusesout of the ‘active site’.

“Lock & Key” (Fischer 1894) and “Induced Fit” (Kosh-land 1958) models are routinely used to explain enzymeactivity. The currently conceived CYP reaction model em-ploys a version of the latter scheme to explain the sub-strate selectivity and the former scheme is invoked toexplain reaction specificity. It is suggested that the F andG loops/helices are considerably flexible across all CYPs(Poulos and Johnson 2005; Narasimhulu 2010). There isalso crystallographic evidence to suggest that CYPs have“closed” and “open” conformations (Poulos 2014). Thisfinding is taken to support the suggestion that a CYP canopen up for a substrate and then close itself upon thesubstrate after its presentation, thereby “committing it tocatalysis” (Lu 1998). The erstwhile hypothetical paradigmis challenged by the fact that CYPs are typically character-ized by broad selectivity/specificity. That is, a given CYPenzyme might catalyze the metabolism of a diverse arrayof substrates of various topographies and dimensions(containing functionally distinct moieties), at multiple lociof the given drug molecule (Sugimoto and Shiro 2012;Ekins et al. 1999; Lewis and Dickins 2002). The crystalstructures of major CYPs are known today, complexedwith some of their known substrates (Williams et al. 2003;Wester et al. 2004; Ekroos and Sjögren 2006; Sevrioukovaand Poulos 2012). In many of these systems, the substrateis positioned too far from the heme-centre for a directattack at the reactive moiety (Ekroos and Sjögren 2006).Also, it is difficult to visualize why/how a constrainedactive site of CYP would not give an enantioselective orregioselective reaction for some large molecules. Further,it is intriguing how some of the most sterically obstructedsites within a given substrate is hydroxylated when thereare other favorable loci available within the very substratemolecule (given the understanding that Compound I, theenzymic reactive intermediate, is supposedly a high poten-tial species). Therefore, some spatial considerations appar-ently challenge the hitherto available ‘highaffinity substrate binding at active site’ theory.A few years back, we had proposed alternative modalities

of substrate interactions for the heme-thiolate (extracellular,fungal) enzyme, chloroperoxidase (Manoj 2006; Manoj andHager 2008). Recently, we have also solved several aspects

Venkatachalam et al. In Silico Pharmacology (2016) 4:2 of 38

of heme-enzyme activations, inhibitions and other kin-etic observations using appropriate biochemical experi-mental controls and logical deductions (Gideon et al.2012; Manoj et al. 2010a; Manoj et al. 2010b; Parasharet al. 2014; Parashar and Manoj 2012). In these works,we went beyond the purely classical substrate-bindingbased Michaelis-Menten kinetics. We wondered if theseideas would be relevant in CYP-drug metabolismmechanistic chemistry. Molecular docking and theoret-ical predictions (based on dataset training/modeling/dynamic simulations) are an efficient and accepted wayof understanding structure-function correlations (Yurievet al. 2015; Cross and Cruciani 2010; Scotti et al. 2015;Hlavica 2015; Olsen et al. 2015; Cruciani et al. 2005;Mudra et al. 2011; Kirchmair et al. 2012; Mendieta-Wejebe et al. 2011; Lewis and Ito 2010). Therefore, weundertook an in silico exploration study of the availablecrystal structures of human microsomal CYPs and probedits “static” docking interactions with diverse “flexible”substrate drug molecules.

MethodsDimensions of small moleculesDimensions of the substrates were calculated usingMarvinSketch 6.2.0 (http://www.chemaxon.com). Theoption of geometrical descriptors was used for this purpose.

Crystal structures of proteins employedTable 1 details the names and references for the pdb filesemployed in the current study.

Cavity analysisCrystal structures of CYPs obtained from RCSB PDBwere visualized using PyMol 1.3 (DeLano 2002) andCAVER 3.0.1 (as a plugin in PyMol) (Chovancova et al.2012) was used to calculate and visualize the tunnels. Allmolecules were analysed with default parameters exceptfor the minimum probe radius (cut off diameter) whichis mentioned against the corresponding entry (Table 2).From the output files, length, curvature and bottleneckdata of the proteins were obtained. Substrates from sub-strate bound protein PDB structure were deleted usingChimera 1.7 (Pettersen et al. 2004) to obtain open andfree tunnels.

DockingCrystal structures of CYPs were obtained from RCSBPDB and used as rigid large molecule receptors. Struc-tures of all the substrates used were obtained fromPubChem and energy minimized using Chimera 1.7.Protein and ligand molecules were primed for dockingusing AutoDock tools (MGL Tools 1.5.4) and docked byAutoDock 4.2 (Morris et al. 2009) to explore the bindingsites of ligands on the protein. Blind docking was carriedout with a grid covering the whole protein for 100 runsto identify putative and unorthodox binding sites insideand outside the active site (represented as Blind Dock-ing or GridB). Refined docking was carried out withinwell-demarcated grid on the heme active site region (thehydrophobic pocket above heme) of a given protein witheach ligand for 100 runs (represented as Centred Dock-ing or GridC). PyMol 1.3 and MarvinView 6.2.0 was usedto visualize the output. The conformers or clusters withthe lowest binding energy for a given ligand were deter-mined. In the two ligand scenarios of drug interactionsstudy, we used CYPs pre-docked with a ligand as a rigidprotein (competitive inhibition for the Enzyme, E; sincewe thought it was unlikely that a bevy of molecules couldhave the lesser probable ES + I binding) and docked itagainst a flexible substrate. RMSD values were calculatedusing Chimera 1.7 and it was noted that ProFit also gavesimilar results.

Homology modellingFor genetic predisposition of drugs study, three dimen-sional models of single amino acid substitution mutationcontaining CYPs were generated using SWISS – MODEL(Arnold et al. 2006) against the respective wild type pro-tein structures as templates. The amino acid sequencecontaining the mutated amino acid was given as input andsearched for templates. From the results, the respective

Table 1 Names and references for enzyme crystal structuresexplored in the current study

S.No.

Enzyme PDB identity Reference(s)

1. CYP1A2 2HI4 (Sansen et al. 2007)

2. CYP2A6 1Z11 (Yano et al. 2005)

3. CYP2C9 1R9O, 1OG2,4NZ2

(Wester et al. 2004;Williams et al. 2003;Brändén et al. 2014)

4. CYP2C19 4GQS (Reynald et al. 2012)

5. CYP2D6 2F9Q (Rowland et al. 2006)

6. CYP2E1 3E6I (Porubsky et al. 2008)

7. CYP3A4 1TQN, 3UA1,2J0D, 2V0M,4K9T, 3TJS

(Yano et al. 2004;Sevrioukova andPoulos 2012; 2013;Ekroos and Sjögren2006)

8. CPO 2CPO (Sundaramoorthy et al.1995)

9. P450cam 2CPP (Poulos et al. 1987)

10. FAB 1GAF (Patten et al. 1996)

11. Estrogenreceptor

3ERT (Shiau et al. 1998)

12. Cellobiohydrolase 1DY4 (Ståhlberg et al. 2001)

13. Avidin 2AVI (Livnah et al. 1993)

14. Glucokinase 3IDH (Petit et al. 2011)


http://www.chemaxon.com

Table 2 Cavity analysis of CYPs by PyMol/CAVER

Enzyme (PDB) Channels by Pymol[with one H2O radius]

Channels by CAVER(cut-off diam. in Å)

Lengtha (Å) Vol. (Å3) Curvature a Bottleneck Dia.a (Å) Classical substratedimensions (Å/Å3)

Amino acids lining the bottlenecka

CYP 1A2 (2HI4) Nil 3 (1.8) 21.6 406 1.19 1.9 Theophylin 4.81, 4.44; 175 F256, N257, Q258, L116, I117, T118,D119, F260, L261

CYP 2A6 (1Z11) 1 + 1 (proximal) 7 (1.8), 1 (2) 18.0 222 1.54 1.99 Coumarin 4.96, 3.68; 127 G102, E103, F118, Q104, D108, R101,A371, A105

CYP 2C9 (1R9O) 2 + 1 (proximal) 15 (1.8), 9 (2), 6 (2.2),3 (2.6), 1 (3.4)

13.0 1457 1.25 3.4 Flurbiprofen 6.99, 3.81; 223 S209, I205, V479, E300, A477, E206,S478, T304, L208

CYP 2D6 (2F9Q) 1 18 (1.8) 7.8 797 1.16 4.3 Bufuralol 7.36, 4.91; 267 F120, T309, V370, A305, L484, R374,p373, V308, S304, G306, C443

CYP 2E1 (3E6I) Nil + 1 (Proximal) 10 (1.8), 3 (2), 2(2.2) 21.9 267 (190 + 77) 1.31 2.28 Chlorzoxazone 5.42, 3.71; 144.02 L368, V364, F478, N367, F207, G479,L363

CYP 3A4 (1TQN) 3 14 (1.8), 4(2), 2 (2.4) 12.7 1508 1.21 2.42 Testosterone 6.67, 4.15; 292.55 Q484, L482, E308, R212, S312, L211

CPO (2CPO) 1 2(1.8), 1 (2.6) 8.7 na 1.14 3.16 CBMS 5.15, 3.12; 118.2 F186, F103, E183, A71, O179, V182

P450cam (2CPP) Nil 5 (1.8) 25.8 na 1.34 1.98 Camphor 4.19, 3.93; 160.86 V247, T181, M184, L180, L200, D182,F98, G243, T185

aDetails of highest ranked tunnel in terms of priority. Minimum probe diameter−1.8 Å. Dim./vol. - Maximal projection radius (Å), Minimal projection radius (Å); Van der Waals volume (Å3) na- not available; Curvature =length/distance, where length is the length of the tunnel (distance from the calculation starting point to the tunnel ending point calculated along the tunnel axis) and distance is the shortest possible distancebetween the calculation starting point and the tunnel ending point

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wild type protein structure with maximum coverage andidentity was chosen for modelling. The output was savedas a PDB file and used for docking, as described earlier.

ResultsDistal heme active site cavities/tunnels of some CYPs andinteractions of the enzyme with substratesTable 2 details the physical dimensions of- active sitecavity, classical substrates and the tunnels from distalheme centre leading to the solvent continuum. Investi-gation with PyMol showed that the highly versatileCYP3A4 has three channels leading to the distal activesite cavity (followed by 2 channels for CYP2C9, anotherversatile liver microsomal CYP), more than any of theother CYPs. With CAVER, all major CYPs (withrelatively higher substrate diversities and greater roles inoverall contributions towards drug metabolism, as exem-plified by 3A4, 2C9, 2D6 and 2E1) gave 10–18 channels(with a water molecule’s diameter as the limiting con-straint). Many of these tunnels were relatively long,twisted or too narrow. One would imagine that suchchannels would not serve significant roles in the move-ment of a bulky substrate molecule to the distal hemecentre. The amino acids lining the bottleneck of theactive site channels were seen to vary significantly acrossthe diverse CYPs. This might suggest little commonalityin mechanisms relating to opening/closing of the chan-nels or F/G helices or loops.Table 3 shows the data for the influence of substrate

molecule on the tunnels leading to the distal active site intwo prominent CYPs. That is- we probed to see what hap-pens to the channels in the “induced fit” substrate-boundCYPs. In CYP 3A4, when the substrate or inhibitor isbound, no channels were available (for a water moleculeto enter the distal active site) (Sevrioukova and Poulos2012). Comparison of the crystal structures of CYPs with-out substrates/inhibitor with (i) crystal structure of CYPbound with substrate/inhibitor and (ii) substrate deletedfrom substrate-bound CYPs gave almost the same data asthat of the former. Similar amino acids are still seen andthe overall conformation is more or less retained. So, thepresence of the substrate does not overtly alter the nativestructure of the protein for the two prominent micro-somal CYPs, as seen from the analysis above. This findingdoes not imply significant changes in tertiary structure viaan induced fit mechanism, as the erstwhile hypothesiswould necessitate to explain outcomes.Table 4 shows the data for blind docking and "active-

site" grid-centred docking of several large drug moleculesubstrates with their CYP counterparts. (The details ofmolecular structure and reaction schema are given inAdditional file 1A, Figure A1A 1 &2.) The blind dockingand heme-pocket grid-centred docking gave different re-sults. The first two entries, Trabectedin and Vinorelbine

are molecules that are metabolized by more than oneCYP. In both these cases, binding at the heme distalpocket is not energetically favorable for CYP2E1. Also,the substrate has a much higher volume than the hemedistal pocket of this CYP (Table 2). In heme distal sitecentred docking, CYP2C9 has very little favorable bind-ing energy, poor orientation and heme-Fe to reactivemoiety distance for Trabectedin. For both these mole-cules, the binding energies were more favorable at alter-nate locations on the protein for the CYPs (2E1 and2C9), as evident with the data for blind-docking. For themaverick CYP3A4, at least four substrates (Trabectedin,Benzoxamino-rifamycin, Tacrolimus and Cyclosporin)did not show a favorable binding energy with hemedistal pocket grid-centred docking, but the same substratesshowed spontaneous binding ability at other locations onthe protein. For CYP3A4, comparable (or slightly better)binding energy was seen outside the heme distal pocket forErythromycin, Teniposide and Itraconazole (in comparisonto the active-site grid-centred docking). These substratesgave a favorable binding at several loci on the protein sur-face, as shown with the blind-docking data. In most ofCYP3A4 examples, the heme-pocket grid-centred dockinggave poor substrate presentation geometries with heme-Feto reactive moiety distance ranging ~ 5 to 22 Angstroms.Considering the molecular dimensions of these substratesvis a vis the heme-distal pocket dimensions (as shown inTable 2), it is difficult to envision the parameters of spatial/topographical recognition that subsequently lead to thebinding or positioning of such diverse large molecules inthe constrained heme distal pockets of the CYPs. A simpleanalysis of the large substrate molecules (as exemplified in1, 4, 11, 12, 16 etc. of Table 4) shows that the reaction locusis many times on rather occluded positions (towards themiddle and not the ends/tips). Such reactive loci may beaccessed by the heme Fe-O species with a major openingup of the protein or inversion of the active pocket, accom-panied by significant conformational flexibility on the sub-strate drug molecules. This would be a low probabilityevent when considering the experimental observation(noted in protein-solution state) that a methyl substitutionon an adjacent carbon renders a heterocyclic nitrogen lonepair ineffective from Type II coordination at the hemecentre (Jones et al. 2011).

Classical marker substrates and their interactions with CYPsWe selected a few well-known high affinity protein-small molecule binding examples as control models ofenzyme-substrate binding. The results for these controlsare given in Table 5 (and the docked images are availablein Additional file 1A, Figure A1B 1–8). Only 3/8 ofthe blind or centred docks showed RMSD values ≤ 2.5Angstroms (with respect to the crystal structure).However, a visual examination shows that in the


Table 3 Effect of substrate on active site channels of CYPs

Enzyme Free enzyme Substrate bound enzyme Substrate bound enzyme with deleted substrate

PDB N Lengtha

(Å)Curvaturea Bottleneck

Dia.a (Å)Amino acidslining thebottlenecka

PDB Substrate N Lengtha



N Lengtha



CYP 3A4 1TQN 13 24.2 1.24 2.24 A305, R212,C442, G306,T309, F304,I301, Heme.

3UA1 Bromoergocrytine

0 - - - - 14 20.4 1.21 1.86 A305, T309,G306, C442,Heme

2V0M Ketoconazole 0 - - - - 18 25.0 1.37 4.08 A305, L482,S119, T309,C442, I301,I369, A370,F304

3TJS @ 0 - - - - 16 13.2 1.10 3.4 Heme, A305,I301, T309,S119, F304

4K9T Desoxy-ritonaviranalog

0 - - - - 24 17.1 1.17 3.8 Heme, A305,T309, F304,I369, C442,I301, G306

CYP 2C9 1OG2 17 15.2 1.34 2.76 T301, A297,L362, G298,G296, C435,Heme

4NZ2 $ 6 22.1 1.29 1.15 D293, N107,F114, V113

9 14.5 1.22 1.03 Heme, A297,L366, T301,L362, C435,V113

N - Number of predicted tunnels; a - details given for channel with highest ranked priority@ - Desthiazolylmethyloxycarbonyl ritonavir, $ - (2R)-N-{4-[(3-bromophenyl)sulfonyl]-2-chlorophenyl}- 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide

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Table 4 Docking of very large substrates to CYPs

S.No.

Substrate Enz. Reaction Dimension/Vol. (Å/Å3)

Blind Docking Centred Docking Ref

Lowest Energy(kcal/mol)

Distance (Å) Interactions Lowest Energy(kcal/mol)

Distance (Å) Interactions Presentation

1 Ecteinascidin/Trabectedin

3A4 N-dealkylation 9.35,6.23;664

−3.90 21.1 ASP217CYS239

+29.90 5.8 LEU438 + (Vermeir et al. 2009;Reid et al. 2002)

2C9 −3.24 25.7 ASP49LEU43

−0.63 19.9 LYS206LEU195

-

2E1 −2.23 25.9 GLN401GLU402LYS422

+1699.52 3.0 THR303 +

2 Vinorelbine 3A4 Deacetylation 9.70,6.72;761

−2.48 20.8 LEU211LYS209VAL240

−3.10 22.1 LEU211LYS209VAL240

- (Kajita et al. 2000;Topletz et al. 2013)

2E1 −3.06 30.1 LEU50 +369.73 13.2 THR303 - (Beulz‐Riché et al. 2005)

3 Tacrolimus 3A4 demethylation 9.48,8.06;787

−2.33 24.6 PRO107a +38.21 8.8 ARG105ARG212SER119

- (Lampen et al. 1995)

4 Benzoxazino-rifamycin (Rifalazil)

3A4 hydroxylation 9.22,8.82;913

−3.16 32.8 ILE473THR471

+99.79 9.0 GLY481SER312LEU438ARG212

- (Mae et al. 1996)

Cyclosporin 3A4 Hydroxylation 12.28,7.91;1218

−1.54 22.6 ARG212ASP214

+4.61 18.9 LYS173PRO485GLU486

- (Kelly et al. 1999;Ohta et al. 2005)

5 Erythromycin 3A4 N- Demethylation 8.91,6.49;729

−2.27 20.8 VAL240ASP217

−1.06 20.7 GLN484LYS173ASP174

- (Wang et al. 2000)

6 Teniposide 3A4 O-Demethylation 10.67,7.02;541

−3.75 13.3 SER437TYR430TYR432PHE435ASN361

−3.44 8.9 ARG212SER119ARG105

(+) (Relling et al. 1994;Julsing et al. 2008)

7 Itraconazole 3A4 Hydroxylation 11.32,6.73;529

−5.31 27.6 PRO227 −4.18 21.1 Val240LYS173TYR307SER311

- (Isoherranen et al. 2004;Templeton et al. 2008)

8 Bosentan 3A4 Hydroxylation 7.60,7.22;478

−2.99 26.5 ARG243CYS239VAL240GLU244

−8.14 10.1 ARG212GLU374ARG375ARG105

- (Dingemanse et al. 2002;Chen et al. 2014)

9 Zafirlukast 3A4 Hydroxylation 8.85,8.36;510

−4.50 25.3 ILE120LYS115

−9.20 14.8 PHE213ARG212

- (Kassahun et al. 2005;Katial et al. 1998)

10 Haloperidol 3A4 Reduction −4.37 11.9 −6.98 5.9 (+) (Kudo and Odomi 1998)

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Table 4 Docking of very large substrates to CYPs (Continued)

7.77,6.05;342

ARG106ARG212

ALA305ARG212

Alfentanil 3A4 Dealkylation 6.94,5.26;401

−3.05 20.7 VAL240LEU211

−6.05 9.6 SER119ARG212

- (Klees et al. 2005;Kharasch et al. 1997)

11 Pranidipine 3A4 De-alkylation 9.27,5.62;401

−5.32 24.0 CYS239ARG243

−9.47 8.3 ARG105ARG375GLU374ARG212ALA370

- (Kudo et al. 1999)

12 Bromoergocriptine 3A4 Hydroxylation 9.96,6.10;550

−4.77 28.3 VAL240a −8.04 10.9 SER119GLU374THR224PHE215

- (Wynalda and Wienkers1997; Sevrioukova andPoulos 2012)

13 Troleandomycin 3A4 N-demethylation 10.70,8.04;781

−2.50 20.9 SER222ARG243

−2.89 18.0 LYS208VAL489GLN484

- (Yamazaki et al. 1996)

14 Ritonavir 3A4 Demethylation 10.44,7.54;585

−1.26 26.9 LYS390 −3.58 19.5 SER312GLN484LEU483

- (Kumar et al. 1996)

Dimensions/volume - maximal projection radius (Å), minimal projection radius (Å); van der Waals volume (Å2)Tacrolimus – 2C9 – GridC – 100th (last) ranked is positioned fully inside hydrophobic pocket with energy→ +46.14, Distance – 9.2 Å. (proved with repeat)Itraconazole – 3A4 – GridB – The 3rd ranked (in the 2nd cluster has better presentation)→ 4.18 (10.2 Å)aNo direct interactions (neighboring amino acids provided), − bad presentation, + good presentation, (+) moderate presentation (not optimal but not facing the opposite end either)

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majority of the cases, both blind and centred dockingidentified the same crypt on the protein as the ligationport (Additional file 1A, Figure A1B). The small mole-cules bound in similar fashion, albeit the bindingenergy being a higher value (that is, a higher negativenumber) in the centred docking. A salient example ispresented in estrogen receptor binding to hydroxyta-moxifen (Fig. 1). Therefore, the blind dockingapproach may be considered as a valid methodologyfor finding out putative binding sites on the protein(Hetényi and van der Spoel 2006) other than the sup-posed “active site of heme distal pocket”. (This consid-eration, definitely, does not address the ‘dimensionalconstraints’ aspect of channels being available for the

substrate to access the distal pocket and the dynamics ofprotein “opening up” or “breathing” in solution state.)It is known that many CYPs have marker substrates. (The

details of molecular structure and reaction schema are givenin Additional file 1A, Figure A1C.) Of the four substratesand CYPs studied for active-site grid-centred docking(Table 6), only two CYP-substrate combinations (2C9-Flur-biprofen & 2D6-Bufuralol) afforded favorable presentationand heme-Fe to substrate reactive moiety distance. This iswhen many CYPs gave better binding energies or presenta-tion at the active site with non-marker substrates. Forexample, CYP3A4 gave better values for Flurbiprofen, thanits natural metabolizer, CYP2C9. Also, both CYP3A4 andCYP2D6 gave better orientations with Chlorzoxazone (at

Table 5 Controls for blind/centred docking

S.No.

Enzyme-Substrate Interactionsin crystalstructure

Docking with the same substrate PDB IDof protein

Overall RMSDe

(Å)Blind Docking Centred Docking


Interactions RMSD(Å)


Interactions RMSD(Å)

1 FAB – Haptena TYR100ARG96HIS35

−3.45c TYR100ARG50HIS35TYR33

5.76 −6.05 TYR100ARG96HIS35ARG50TYR33

5.06 1GAF 5.91

2 Estrogen receptor -Hydroxytamoxifen

GLU353ARG394

−7.19 GLU353ARG394

6.61 −8.08 GLU353ARG394

1.70 3ERT 1.19

3 Cellobiohydrolase –INPb

GLU212GLN175GLU217

−3.49 TYR145TYR171SER365

1.40 −4.66 TYR145ARG107

7.82 1DY4 0.99

4 Avidin - Biotin SER16SER73SER75THR35THR38THR40ASN12ASN118

−6.30 SER16SER73SER75THR35THR38THR40ASN12ASN118ALA39

1.36 −7.25 SER16SER73SER75THR35THR38ASN12ASN118ALA39

1.14 2AVI 1.26

5 Glucokinase - Glucose ASN204ASN231ASP205GLU290GLU256GLN287THR168LYS169

−1.66 GLU27GLU28LYS31

31.07 −5.46 ASN204ASN231ASP205GLU290GLU256GLN287THR168LYS169SER151

2.82 3IDH 22.11

6 P450cam - Camphor TYR96 −6.51 TYR96 2.42 −6.93 TYR96 2.50 2CPP 2.42

7 CYP2C9 - Flurbiprophen ASN204ARG108

−5.85d ARG108 16.13 −6.59 ASN204ARG108

5.06 1R9O 11.95

8 CYP3A4 - Erythromycin SER119 −4.48 SER119 7.02 −6.43 SER119PHE304ARG106GLU374

5.98 2J0D 4.97

a5-(Para-nitrophenyl phosphonate)-Pentanoic acidb1-(Isopropylamino)-3-(1-Naphthyloxy)-2-PropanolcData of 2nd cluster provided. Free energy of first cluster is−3.56 kcal/mol (0.11 kcal/mol higher)dData of 4th ranked binding in the 1st ranked cluster. Lowest free energy is−5.86 kcal/mol (0.01 kcal/mol higher)eValue obtained by comparing all the three- crystal structure, blind-docked and centred-docks


comparable binding energies) than its natural catalystenzyme, CYP2E1. These findings indicate that theselection or reaction process need not obligatorilyinvolve binding/catalysis centred in the heme distalpocket alone.Therefore, a detailed blind docking study was carried out

for eight substrates across six major CYPs. In blind docking(Table 7), most of the classical substrates did not afford a

highly fruitful binding at the heme centre. Several drugmolecules afforded better binding or presentation at a non-specific CYP heme-distal pocket. Therefore, with the erst-while understanding, we are at a disadvantage to accountfor the non-reactivity of these substrates with the “non-spe-cific CYPs”. Further, interactions with key amino acids(which could be an argument for specific molecular triggersinvolved in an induced fit type of process) were not seen to

Table 6 Marker Substrates and CYPs – Distal heme pocket-centred docking

Substrate → Flurbiprofen Bufuralol Chlorzoxazone Testosterone

Dim;Vol (Å/Å3) → 6.99, 3.81; 223 7.36, 4.91; 267 5.42, 3.71; 144 6.67, 4.15; 293

2C9 (1R9O) X 4.5 15.2 3.1 9.2

E −6.64 −5.73 −4.63 −7.88

I ARG204 ARG108 LEU208 ALA297 LEU208

P + - + -

2D6 (2F9Q) X 4.8 4.3 3.2 10.2

E −5.81 −6.13 −5.37 −7.33

I PHE483 LEU484 GLU216a ALA305 ALA305 LEU213

P + + + -

2E1 (3E6I) X 9.3 6.8 8.2 8.4

E −2.13 −3.52 −5.49 +11.67

I PHE478 PHE116a ALA299 ALA299 THR303

P - + (+) -

3A4 (1TQN) X 3.3 6.5 5.0 10.0

E −8.03 −6.61 −5.64 −6.61

I GLU374 ARG375 ARG440 SER119 SER119 ARG212

P + + + -

Dimensions/volume - maximal projection radius (Å), minimal projection radius (Å); van der Waals volume (Å3); aneighboring amino acids provided, X – distancebetween heme centre and reaction site (Å). E - lowest binding energy (kcal/mol), I – Interactions. - bad presentation, + good presentation, (+) moderatepresentation (Not optimal but not facing the opposite end either)

Fig. 1 Docking (blind and heme-distal pocket centred) of hydroxytamoxifen to estrogen receptor and comparison with the crystal structure. The RMSDvalues are given in Table 5


Table 7 Marker Substrates and CYPs – Blind docking

Substrate(CYP preference)→ Theophylin (1A2) Diclofenac (2C9) Warfarin (2C9) Flurbiprofen (2C9) Mephenytoin (2C19) Bufuralol (2D6) Chlorzoxazone (2E1) Testosterone (3A4)

Dim;Vol (Å/Å3) → 4.93, 4.35; 147 6.00, 4.82; 240 6.54, 5.28; 277 6.99, 3.81; 223 5.66, 4.46; 201 7.36, 4.91; 267 5.42, 3.71; 144 6.67, 4.15; 293

1A2 (2HI4) X 7.0 32.3 24.1 21.1 32.0 32.4 4.7 20.0

E −4.16 −4.52 −4.47 −5.39 −3.14 −2.55 −4.55 −4.78

I THR124 LYS277LYS292LYS293

PRO84CYS406LYS404SER389ASP110

TYR495LYS59ASN60

LEU51ILE241

LEU242a THR124 TRP466ARG362

2C9 (1R9O) X 19.8 18.8 23.7 17.3 11.0 15.2 7.7 20.2

E −3.34 −6.15 −5.72 −5.73 −4.31 −4.43 −3.96 −6.48

I PHE100 LYS72 THR364 LYS72PHE100

ALA297a SER209a

LEU208aTHR301a

LEU366aPHE100THR364

2C19 (4GQS) X 6.0 20.7 4.6 4.1 8.8 6.0 5.2 7.7

E −3.65 −4.38 −5.44 −4.87 −5.11 −4.82 −4.37 −6.90

I GLY296 LYS270 ILE205a THR301a GLY296 LEU202a LEU366a

GLY296aVAL113a

2D6 (2F9Q) X 7.4 17.3 12.8 20.6 10.5 6.6 7.0 19.6

E −4.17 −5.59 −5.17 −5.25 −4.57 −4.28 −5.06 −6.48

I ALA305 HIS178TYR56

SER217a HIS478GLY479

LEU213a LEU484a

LEU213aALA305 HIS478

2E1 (3E6I) X 6.5 28.3 4.9 17.8 30.6 29.3 27.6 18.2

E −3.51 −4.79 +4.52 −4.83 −3.81 −3.86 −4.12 −5.11

I PHE298a ARG379LYS84

LEU210a LYS408 LYS486 ASP470ARG484

LYS486LEU463

THR58ILE361

3A4 (1TQN) X 5.3 3.5 10.8 10.6 8.3 4.2 4.7 12.8

E −4.51 −5.77 −4.79 −5.27 −4.10 −4.45 −3.96 −6.10

I ALA370a ARG105 ARG105 ARG212 ARG212 ARG105ARG212

ARG105 SER119ARG212

GLY436PHE435

X – distance between Fe and reaction center (Å), E – lowest energy (kcal/mol), I – InteractionsDimensions/volume - maximal projection radius (Å), minimal projection radius (Å); van der Waals volume (Å3)Diclofenac – 2C19 the 2nd cluster (9th ranked) is inside active site (−4.35, 4.8 Å)Testosterone – 2C9 the 3rd cluster (77th ranked) is inside active site (−5.90, 5.9 Å)Testosterone – 3A4 the 2nd cluster (39th ranked) is inside active site (−5.82, 9.3 Å)Flurbiprofen – 2C9 the 3rd cluster (56th ranked) is favorably inside active site (−5.33, 4.1 Å)Theophylin – 1A2 the 2nd cluster (31st ranked) has favorably inside the active site (5.1 Å, −4.04)ano direct interactions (neighboring amino acids provided)

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be critical either, in making or marking a molecule aspotential substrate. In silico binding of Warfarin to 2C9,testosterone to 3A4, etc. afforded results that disagreed withcrystal structures whereas docking of Flurbiprofen to 2C9,Bufuralol to 2D6, etc. agreed with the crystal structures.(These findings are in agreement with our earlier works,Parashar et al. 2014). Of the combinations tested, onlyCYP2C9 and CYP3A4 (both possessing large distal pocketsof ~1500 cubic Angstroms; rendering the heme distalpocket less consequential with respect to spatial constraints)gave similar ranking results for both blind and heme-distalpocket grid-based dockings for their marker substrates.

Docking of select classes of drug molecules (Sartans,Statins & Triptans) with CYP2C9 and CYP3A4Table 8 shows the docking of different Sartans (Thedetails of molecular structure and reaction schema aregiven in Additional file 1A, Figure A1D.) to theCYP2C9 pdb files 1R9O and 1GO2 respectively. It isknown that Irbesartan and Losartan are efficient sub-strates, Candesartan and Valsartan are poor substratesand Tasosartan and Olmesartan are not substrates ofCYP2C9 (Kamiyama et al. 2007; Berellini et al. 2005;Perrier et al. 1994; Sica et al. 2005; Stearns et al. 1995).Binding of Sartans (that possess a relatively large andwell presentable pharmacophore, which contributedto ≥ 1/2 the surface area/volume of the whole drugmolecule) with 1R9O were seen to be at different lociin blind and heme-distal site centred dockings. In blinddocking, the Sartans always bound with better energyterms outside the heme distal pocket (Additional file1A, Figure A1E). When considering centred binding atthe distal heme pocket, all Sartans (substrates or non-substrates) bind in a similar fashion, with comparableenergies and orientation of reaction sites. (The image isshown in Additional file 1A, Figure A1E) In these cases(and also in blind docking), the heme-Fe to reactioncentre on the substrate distance is not conducive fordirect oxygen transfer. The Sartans and/or its derivativepossessing a carboxy moiety on the R-group were eitherinefficient substrates or efficient inhibitors of CYP2C9mediated metabolism. Since these molecules bind atidentical loci (interacting with the same amino acids)within the heme pocket, the difference in substratereaction or inhibition potency is difficult to beexplained merely by an active site binding hypothesis.More importantly, rather than the active site position-ing, substrate reactivity per se and also the interfacialROS (reactive oxygen species) modulation by suchmolecules could explain the outcomes (Parashar et al.2014). With 1GO2, 6/8 of the blind docking andcentred docking gave similar clusters. Though bindingenergies are more favorable in the heme distal pocketcentred docking, the distances are still too high to

explain for activities. Once again, distal heme-pocketbinding mechanism is inadequate to explain the re-activity or specificity of Sartans with this structure also.The six chosen Statins had two different small

pharmacophore groups, as shown in Additional file 1A,Figure A1F. It is known that Fluvastatin and Pravastatinare efficient substrates, Lovastatin is poor substratewhereas Mevastatin, Simvastatin and Atorvastatin arenot metabolized by CYP2C9 (Neuvonen et al. 2008;Bellosta et al. 2004; Chapman and McTaggart 2002). Thepharmacophore posed ≤ 1/2 area/volume contribution tothe whole molecule. The results of docking these mole-cules to CYP2C9 (1R9O) are shown in Table 9 (and thecentred docking image is shown in Additional file 1A,Figure A1G). Two drugs with the common flurophenylpharmacophore, Fluvastatin (substrate) and Atorvastatin(not a substrate), bind at similar locus in heme-pocketcentered docking (though presentation of reaction centrevaries), when the most energy-minimized conformationsare taken. The second most preferred binding conformer(w.r.t. binding energy) of Atorvastatin presents favorablynear the reaction centre, at a comparable energy term(as required by Fluvastatin to achieve the same distancefrom reaction centre). Therefore, the reactivity of thesetwo statins is not explained by heme-pocket substratebound reaction model. On the other hand, blind dockinggave very different energy values for these two Statins(with much lesser affinity for Atorvastatin in comparisonto Fluvastatin) and they were found to bind at differentloci on the protein. The four Statins with bicyclohexenylpharmacophores could be graded into three classes-Pravastatin (good substrate), Lovastatin (weak substrate)and Mevastatin/Simvastatin (not substrates). The hydro-phobicity of these Statins increase in the order 2 < 4 < 3< 5. The binding energies decrease (as expected) and dis-tance of reactive moiety from the iron centre becomemore favorable for these four statins in the same corre-sponding order. Of these, three interact with the sameamino acid(s) at the active site, although with similarorientation of the pharmacophore and presentation ofthe reaction centre. Simvastatin (the most hydrophobicof the Statins, not a substrate), afforded the most prox-imal orientation of the reaction centre (4.8 Å), whencompared to the value of 10.6 Å for the substrate Prava-statin (the least hydrophobic of the statins). Mevastatin,though more hydrophobic than Pravastatin, can ap-proach the heme centre at 4.7 Å (for the 10th rankedconformer, with a binding energy of ~ −7.5 kcal/mol),but yet, it is not a substrate. Therefore, once again, “thesubstrate binding the heme-distal pocket followed by re-action” model falls short at explaining the reactivity ofthe Statins. In blind docking, the four Statins were foundto bind at different loci on the protein (as shown inimage Additional file 1A, Figure A1H).


The docking studies with Sartans and Statins showthat although blind dockings of some substrates gavesimilar results when compared to heme-distal pocketcentred dockings, yet other substrates gave quite differ-ent docking sites in the blind docking. Also, for a givenCYP, several non-substrates were found to have compar-able binding energies (and even better relative orienta-tions) to substrates. Further, it is difficult to visualizehow a molecule like Atorvastatin could ever get to beoxidized by CYP3A4 at the ortho (and not para) positionon the terminal phenyl ring (Additional file 1A Figure

A1F), if the reaction were to occur at the spatially con-strained heme centre.Triptans, possessing a central indolyl pharmaco-

phore, were chosen as a probe for CYP3A4 and the re-sults are shown in Table 10. (The details of molecularstructures and reaction schema are given in Additionalfile 1A, Figure A1I.) Only Eletriptan, Almotriptan andNaratriptan are known to be substrates of CYP3A4(Evans et al. 2003; Salva et al. 2003; Sternieri et al.2006; Moore et al. 2002; Wild et al. 1999; Vyas et al.2000). Blind and centred docking gave different

Table 8 Sartans-CYP2C9

No. Substrate Reaction Dimension/Vol. (Å/Å3)

CYP2C9-1R9O top row; CYP2C9-1GO2 bottom row

Blind Docking Centred Docking


Distance(Å)

Interactions Lowest Energy(kcal/mol)

Distance(Å)

Interactions Presentation

1 Irbesartan Hydroxylation 8.11,6.21;397

−5.60 24.0 LYS72a −8.62 4.3 ARG108 +

−6.86 5.2 LEU208ASN474GLN214PHE476

−7.63 3 GLY98PHE100

+

2 Losartan Oxidation 7.77,5.43;372

−4.59 24.3 PHE69a

PRO221a−7.98 10.0 GLU104a -

−5.57 10.8 GLY98,GLY296PHE100

−7.00 9.9 GLY98PHE100

-

3 Exp – 3179 Oxidation 8.22,5.67;322

−6.92 17.1 LYS72a −8.35 10.0 ARG108a -

−6.01 10.7 ASN474GLN214LEU208GLY296

−6.88 10.1 PHE476THR301

-

4 Exp – 3174 Inhibitor 8.14,5.61;329

−6.97 26.1 PHE100PHE69LYS72

−8.64 10.8 LEU208ARG108

-

−5.73 13.8 PHE100 −5.78 7.7 THR301LEU208ASN474GLN214

(+)

5 Candesartan O- Deethylation 8.24,5.39;383

−7.22 16.6 PRO221a −8.40 10.5 LEU208 -

−5.09 7.0 GLY98ASN107GLY296PHE100

−6.34 8.3 GLY98 (+)

6 Valsartan Hydroxylation 8.43,5.94;410

−5.65 20.7 LYS72 −7.31 8.4 ARG108 (+)

−4.79 23.7 LYS273 −6.47 7.3 GLY98PHE100

(+)

7 Tasosartan (CYP3A4 substrate) 8.59,5.32;362

−8.00 nk PRO221 −9.25 nk LEU208 nk

−6.65 nk PHE100 −8.53 nk PHE100 nk

8 Olmesartan Not substrate 8.47,5.96;404

−5.26 nk LYS72PHE69

−8.15 nk LEU208 nk

nk LEU208THR301ASN474

−6.35 nk LEU208THR301ASN474GLN214

nk

Dimensions/Volume - maximal projection radius (Å), minimal projection radius (Å); van der Waals volume (Å3); For 7 & 8, reaction site not known. - bad presentation, +good presentation, (+) moderate presentation (not optimal but not facing the opposite end either); a - no direct interaction, neighborring amino acids provided


Table 10 Triptans-CYP3A4

S.No.

Substrate Reaction Dimension/Vol. (Å/Å3)

CYP3A4 (1TQN)



Distance(Å)


Distance(Å)


1 Eletriptan N-demethylation 6.05,5.44;355

−4.99 17.4 CYS239ARG243

−7.82 7.6 ARG105SER119ALA305

-

2 Almotriptan N-demethylation 6.03,5.56;313

−4.00 17.5 CYS239ARG243PHE241

−6.66 7.4 ARG372ARG105

-

3 Naratriptan N-Demethylationa 7.70,5.52;312

−4.66 17.3 CYS239ARG243

−5.52 10.4 GLU374ARG106

-

4 Sumatriptan N-Demethylationa 6.25,5.30;232

–4.39 17.7 CYS239ARG243PHE241

−3.77 8.1 ARG106ARG212

-

5 Zolmitriptan N-Demethylation(CYP1A2)

5.59,5.05;273

−4.28 17.9 CYS239ARG243

−6.24 10.8 ILE369GLU374ARG375ARG105

-

6 Rizatriptan N-Demethylationa 6.17,5.56;259

−3.64 16.5 CYS239 −5.62 7.9 ARG372SER119

-

Dimensions/Volume - maximal projection radius (Å), minimal projection radius (Å); van der Waals volume (Å3). - bad presentationaN-demethylation is assumed as the reaction centre

Table 9 Statins-CYP2C9

No. Substrate Reaction Dimension/Vol.(Å/Å3)

CYP2C9-1R9O



Distance(Å)


Distance(Å)


1 Fluvastatin Hydroxylation,C5-, C6-

6.94,6.24;383

−5.05 19.6 PRO221LYS72

−6.80 7.4 ARG108ASN204

(+)

2 Pravastatin Hydroxylation, C3’- 7.65,6.25;415

−5.39 20.0 SER53 −7.35 10.6 ARG108ASN204GLY296ALA297

-

3 Lovastatin Hydroxylation,6’beta-, C3’-, C5’-

8.19,5.64;405

−6.63 19.6 LYS72 −8.18 10.4 ASN204VAL292

(+)

4 Mevastatina Hydroxylation, C3” 7.06,5.42;385

−6.65 21.0 LYS72 −7.84 10.4 ARG108 -

5 Simvastatina Hydroxylation,C3’-, C5’-

8.08,6.34;422

−6.68 4.9 SER209ASN474THR304

−8.80 4.8 SER209ASN474THR304

+

6 Atorvastatina Hydroxylation,C2-, C4-

7.08,6.62;517

−2.55 29.1 LYS206ASN474PHE482

−5.28 17.9 ARG108 -

Dimension /Volume- maximal projection radius (Å), minimal projection radius (Å); Van der Waals volume (Å3)- bad presentation, + good presentation, + moderate presentation (not optimal but not facing the opposite end either)The second preferable binding for Atorvastatin is −4.15 kcal/mol, with 3.8 A from iron center, once again binding to Arg 108. For Fluvastatin to achieve the samedistance from the reaction center, a binding energy of −5.78 kcal/mol was notedaMetabolized by CYP3A4; underlined numericals have more structurally similar pharmacophores


binding locations for the Triptans. In the heme distalpocket-centred docking, a substrate is bound in thesame manner as a non-substrate. Presentation is notfavorable in the active site, with more than 7 Å dis-tance in each. In blind docking, all Triptans bind to aconserved but different locus within the heme-distalpocket. Neither binding energy analysis nor substrateproximity/orientation (within the heme distal pocket)could afford a convincing explanation for Triptansubstrate preferences or reactivity for CYP3A4.

Demarcating substrate preferences between related andunrelated CYPsTable 11 shows the blind and centred docking results forOmeprazole binding with two highly related CYPs- 2C9and 2C19. Omeprazole is a substrate for CYP2C19, butnot of CYP2C9 (Äbelö et al. 2000). In the heme-pocketcentred docking, both 2C9 and 2C19 gave similarlybound substrate molecules, with comparable binding en-ergies and orientations. [The docking results (images)are given in Additional file 1A, Figure A1J 1 & 2.] Theblind docking gave different Omeprazole binding loci onthe proteins (which showed a greater proximity ofOmeprazole to the heme centre in CYP2C19), whichcould tentatively explain the preference of CYP2C19 forthe substrate.Table 12 shows the result for the in silico binding of

various oxyresorufins with two relatively unrelatedCYPs- 1A2 & 3A4. For 1A2, alkyloxyresorufin is thepreferred substrate, with -C2H5 being the optimalside-chain and much lower activities being observedwith larger or smaller substitutions. On the otherhand, 3A4 shows preference for an aryl substitutionand little activity with smaller/linear chain substituent(Kenworthy et al. 1999). The heme-pocket centreddocking results indicate that enhancement of substitu-tion bulk affords better binding energy terms withoutany significant alteration of the substrate bindinglocus and presenting modalities, for all substrates, inboth 1A2 & 3A4. The centred docking shows that allfour substrates are poorly presented to 1A2. In com-parison, 3A4 is seen to bind all four substrates at theactive site in relatively favorable modes [Images ofthese docking results are given in Additional file 1A,Figure A1K 1–4]. Therefore, the heme-distal pocketbinding-based reactivity cannot adequately explain the

preference of ethoxyresorufin by 1A2 and benzyloxyr-esorufin by 3A4. In contrast, changing the substituentalters the binding locus and modalities in both 1A2and 3A4 for blind dockings, thereby offering scope forexplanation of kinetic preference of substrates.

Revisiting the classical mutation experiments of RLPLindberg & M Negishi with the crystal structures ofCYPs- 2A6 & 3A4Coumarin is the natural substrate for CYP2A6 andtestosterone is for CYP3A4 (Yun et al. 1991; Wang et al.1997). It was seen in a pioneering study in 1989 thatmutating a single residue (P209L) in CYP2A6 changed itto a testosterone hydroxylating enzyme, akin to CYP3A4(Lindberg and Negishi 1989). Table 13 reports the dock-ing data for probing these salient observations.For coumarin, both docking grids gave similar results

in the two wildtype and the two mutant CYPs. That is-coumarin bound in the same locus in these enzymes(within the distal pocket) regardless of the modalityemployed for docking or mutations made (Table 13)[The docking results (images) are given in Additionalfile 1A, Figure A1L 1–4]. Therefore, the hithertoconsidered mechanism fails to explain the loss/gain ofactivity when there is an extremely similar binding ofcoumarin inside the heme distal pocket in the wild typeand mutated 2A6/3A4. If the presentation and bindingof substrate at the heme distal pocket were to be cru-cial, then testosterone should be a good substrate forCYP2A6. In CYP3A4, the high loss of activity observedwith mutation of Leu 209 (the crystal structure Leu210) to Phe 209 cannot be explained by a heme-distalpocket centred binding. Leu 209 is ~14 Å away fromthe heme (and ~7 Å away from the distal pocket chan-nel at the closest locus) and is more closer to the sur-face (part of the amino acid residue can be visualizedlocated in a small crypt on the surface; (Additional file1A, Figure A1L 5 and 6) than to the heme floor. It doesnot form the entrance of any of the three major chan-nels that lead to the distal cavity. Further, it is difficultto imagine testosterone presenting itself in the hemedistal pocket in a suitable manner (to undergo oxygenrebound at the heme centre), unless there is a large-scale opening up of the distal site. The two mutations(Ala 117 to Val 117 & Leu 365 to Met 365) that affectboth coumarin and testosterone activities of CYP3A4

Table 11 Omeprazole – 2C9 and 2C19

Substrate Enz. Blind docking Centred docking

Lowest Energy (kcal/mol) Distance (Å) Interactions Lowest Energy (kcal/mol) Distance (Å) Interactions Presentation

Omeprazole 2C9 (1R9O) −5.31 19.9 PRO37 −6.99 17.1 (2.5) LEU208 -

2C19 (4GQS) −4.43 12.8 ALA206 −6.51 10.5 (2.6) ASN107 -

- bad presentation


are stuffed into the protein core, located at ~4 Å and~10 Å respectively (at the most proximal loci with re-spect to the distal pocket/channel). In blind docking,testosterone binds to CYP3A4 at a crypt adjacent toLeu 209, with a binding energy of − 4.7 kcal/mol. Ifbinding of the substrate to the surface was importantto catalysis, we could explain the mutation’s outcomefor CYP3A4. Significant loss of activity for coumarinis seen in CYP2A6 only with a simultaneous alter-ation of all three residues, Val 117, Phe 209 & Met

365. These resides are found on different loci withinthe protein (different helices/loops). Met 368 (perhapsthe same as Met 365 in the earlier literature) is faraway from the channel that leads to the heme. Hemepocket - centred docking of testosterone with thehomology models (with the mutated residues) gaveslightly different results, when compared to the wildtype. But these slight differences in binding energyand distance also fail to explain the complete reversalof activity of 2A6 and 3A4.

Table 13 Coumarin/Testosterone : CYP2A6 (1Z11) & CYP3A4 (1TQN)

Substrate Enz. Blind docking Centred docking




Coumarin 2A6 −5.83 5.4 ASN297 −6.33 5.1 (2.8) ASN297 +

m2A6 −5.83 5.6 ASN297 −6.16 6.2 (4.3) ASN297 LEU296 -

3A4 −5.42 5.0 ARG212 SER119 −5.70 4.7 (3.5) ARG212 SER119 +

m3A4 −5.11 4.8 ARG212 SER119 −5.65 4.7 (3.5) SER119 +

Testosterone 2A6 −4.83 35.6 LYS32 −5.58 8.0 (3.0) PHE107 -

m2A6 −4.68 32.1 PRO231 GLN234 −7.83 8.4 (2.9) MET205 +

3A4 −6.11 12.8 GLY436 PHE435 −6.61 10.0 (4.5) ARG212 -

m3A4 −5.95 12.8 GLY436 PHE435 −6.56 11.7 (7.8) ARG105 GLU374 ARG375 -

- bad presentation, + good presentationm2A6 (P209L) – Test – GridB - 82nd and 96th ranks bind near the mutated residue2A6 – Test – GridB – 87th to 89th ranks bind near the amino acid under studym2A6 (P209L) – Cou – GridB - 7.8 Å away in 1st rank2A6 – Cou – GridB – 4.8 Å from 1st rankm3A4 (L210P) – Cou – 90th to 98th ~ 11 Å from the mutated residuem3A4 – Cou – 90th to 100th ~ 11 Å from the mutated residuem3A4 (L210P) – Test – 42nd to 95th bind around 7 Å away from the mutated residue.m3A4– Test – 50th to 99th ranks bind with ~ 9 Å away from the mutated residueData given in braces – nearest distance between any atom of ligand and heme center

Table 12 Oxyresorufins- 1A2 and 3A4

Enz. (pdb) Substrate Blind docking Centred docking




1A2 (2HI4) Methoxy-resorufin −6.02 4.5 THR124 −6.86 11.3 THR124 -

Ethoxy-resorufin −5.96 11.3 THR124 −7.15 11.3 THR124 -

Pentyloxy-resorufin −5.91 11.6 THR124 −8.05 11.5 THR124 -

Benzyloxy-resorufin −4.28 34.0 PHE239b −8.20 11.5 THR124 -

3A4 (1TQN) Methoxy-resorufin −6.86 18.0 PHE435GLY436

−5.67 4.2 ARG105GLU374ARG375

+

Ethoxy-resorufin −4.22 9.2 ARG212ARG105GLU374

−6.11 4.2 SER312ILE369LEU483

+

Pentyloxy-resorufin −4.57 9.6 ARG105GLU374ARG212

−6.60 4.5 ALA370GLU374ARG375ARG105

+

Benzyloxy-resorufin −5.45 6.7a ARG212 −7.44 4.0 SER312LEU483

+

- bad presentation, + good presentationa- 3.8 Å for phenyl ring; b - no H-bonds or pi-stacking interactions (neighboring amino acids provided)


Genetic predispositionsIn the first study, allelic variation of 2C9 activity onBosentan was studied. The alleles 2C9*13 (L90P) and2C9*43 (R124W) had very low Bosentan clearance (<1 %of control) whereas 2C9*55 (L361I) has very high activity(Chen et al. 2014). It should be remembered thatBosentan is a very large substrate and it poses littlescope for the direct presentation of its reactive moiety atthe iron centre of distal heme pocket. Homology modelsof the proteins were prepared and it was seen that noneof the amino acid substitutions caused a conformationalchange in the heme distal pocket region or the overallstructure of the protein. The variant 2C9*13 (with sig-nificantly reduced intrinsic clearance values) had a morefavorable binding energy and distance in both blind andgrid centered docking (Table 14). In the case of 2C9*43,the slight increase in the distance inside the heme distalpocket does not explain the very low activity seen. In2C9*55 (the allele with highest Bosentan activity), theleast favorable binding (in terms of distance and presen-tation) inside the active site gave the highest activity.This is yet another indication that a heme-pocket bind-ing based reaction mechanism is inadequate to explainthe differences in activity seen in any of the allelesstudied.In the second study (5, 6 & 7 of Table 14), the allelic

variation of CYP2C9 activity on the marker substrateWarfarin was studied. The alleles 2C9*3 (R125H) and2C9*16 (T299A) have very low Warfarin clearance(DeLozier et al. 2005). The heme-pocket grid centreddocking of the wild type and the homology modelledalleles gave almost exact bindings in all rudiments. Onceagain, the hitherto perceived mechanism does notexplain these results.

Drug-drug interactionsIn Table 15, Grid 1 refers to blind docking of the sub-strate with the protein bound to the top-ranked con-former of modulator from its blind docking. Grid 2

refers to blind docking of the substrate with the proteinbound to the nth ranked modulator, which coincideswith the substrate’s (lone presentation) binding locus.Overall, 20 instances of drug interactions (as reported inliterature, with particular reference to the data furnishedby the groups of Houston (Kenworthy et al. 1999), Tracy(Wang et al. 2000) and Birkett (Miners and Birkett1998) were investigated and the pertinent results areshown in Tables 15 and 16. The structures of the con-cerned molecules (substrates and modulators) are shownin Additional file 1A, Figure A1M. Nine of these led toinhibitions, nine were instances of activations and twocases were concentration-dependent, leading to eitherinhibitions or activations. Most importantly, it could beseen that a molecule like quinidine could activate anisozyme like CYP3A4 (10, when Meloxicam is thesubstrate), inhibit the same isozyme (6, when Nifedipineis the substrate) and activate or inhibit the very isozymedepending upon its concentration (8, when testosteroneis the substrate). Further, quinidine could showconcentration-dependent effects across CYPs (7 for 2C9& 8 for 3A4). At the outset, such effects are very difficultto be intuitively or logically accounted for by a purelyactive site or allosteric binding-based phenomenon.Also, Occam’s razor would suggest that such mechanis-tic processes would have little significance for physio-logical evolution, for an active site to develop suchintricate patterns of activities.From Table 15, it can be noted that constrained dock-

ing of the modulator at the distal pocket does not pre-vent the secondary binding of any of the substrates atthe active site (except the case 2). We see that inhibi-tions are not explained with the heme-pocket bindinghypothesis because in certain cases (as exemplified by 1and 2), the distance between heme-Fe and the reactioncentre decreases upon the presence of modulator.Further, in examples like 1 & 5, binding energy for thesubstrate becomes more favorable with the presence ofmodulator. Regarding activations and a concentration-

Table 14 Genetic predisposition of drug metabolism

No Enzyme/Allele

Mutation %Activity

Substrate Docking



Distance(Å)


Distance(Å)


1 CYP 2C9 Wild type 100 Bosentan −2.67 27.9 LEU222 −6.40 3.1 ASN217 +

2 2C9*13 L90P 0.92 −4.75 5.1 PHE100 −6.43 3.0 ASN217 +

3 2C9*43 R124W 0.55 −2.52 29.6 LEU222 −6.37 4.0 PHE100 +

4 2C9*55 L361I 488.89 −2.95 13.4 LEU208 −6.37 11.7 THR301 -

5 CYP 2C9 Wild type 100 S - Warfarin −5.01 11.1 ASN217 −7.28 10.7 PHE100 ASN217 -

6 2C9*3 R125H 7 −4.99 12.6 PHE100 −7.19 10.8 PHE100 ASN217 -

7 2C9*16 T299A 8 −4.48 12.3 THR301 −7.18 10.8 PHE100 ASN217 -

- bad presentation, + good presentation


Table 15 Drug-drug Interactions

S.No.

Enzyme + boundligand

Substrate Blind docking Centred Docking

Two-ligand scenario Substrate alone

Grid1 Grid2 Two-ligand scenario Substrate alone

E (kcal/mol)

X (Å) Interactions E (kcal/mol)




X(Å)

Interactions

1 2C9.Fluvastatin Diclofenaca −4.94 9.5 ARG108ASN204

NA −6.15 18.8 LYS72 −6.79 7.4 ARG108ASN204

−6.39 17.0 ARG108

2 3A4.Clotrimazole Erythromycin −2.15 20.2 VAL240 −2.19 17.3 LYS209ARG212ASP214

−2.27 20.8 VAL240ASP217

+16.85 18.0 GLN484SER312SER311SER315VAL489

−1.06 20.7 GLN484LYS173ASP174

3 3A4.Itraconazole Testosterone −6.04 12.8 GLY436PHE435

−5.50 9.2 ARG105 −6.10 12.8 GLY436PHE435

−5.65 16.1 GLN484TYR307

−6.61 10.0 ARG212

4 3A4.Ketoconazole Testosterone −6.09 12.7 GLY436PHE435

−5.71 10.3 ARG105 −6.10 12.8 GLY436PHE435

−6.33 15.9 GLN484SER312TYR307

−6.61 10.0 ARG212

5 2C9.Fluconazole Warfarin −5.54 24.2 TRP212 −5.56 28.8 LYS72ILE223

−5.72 23.7 THR364 −8.32 18.0 GLU104LEU102

−6.34 12.9 THR301GLY296

6 3A4.Quinidine Nifedipinea −3.97 24.3 CYS239PHE241ARG243GLU244

NA −4.83 9.7 SER119 −4.45 11.7 SER119 −6.55 7.9 ARG212

7 2C9.Quinidine Meloxicam −6.10 14.3 LEU208 −5.92 30.1 THR364 −5.82 14.4 LEU208 −7.19 4.2 ASN204GLY296

−6.97 15.3 LEU208

8 3A4.Quinidine Testosterone −6.09 12.8 GLY436PHE435

−5.92 24.3 THR364 −6.10 12.8 GLY436PHE435

−6.14 12.2 GLU374ARG372

−6.61 10.0 ARG212

9. 2C9.Dapsone Flurbiprofena −5.67 5.0 ASN204ARG108

NA −5.73 17.3 LYS72PHE100

−7.19 17.2 ARG108ASN204

−6.64 4.5 ASN204ARG108

10. 3A4.Quinidine Meloxicam −5.32 9.7 ARG212PHE108GLU374ARG372

−4.38 21.0 ARG243PHE241VAL240CYS239

−4.45 10.2 ALA370 −8.53 10.2 PHE213SER119

−6.95 12.7 SER119ARG212ARG105

11. 3A4.Hydroquinidine Meloxicama −4.38 20.1 CYS239PHE241ARG243VAL240

NA −4.45 10.2 ALA370 −9.94 10.1 ARG212 −6.95 12.7 SER119ARG212ARG105

12 3A4.Budesonide Dextromethorphan −4.42 18.6 ASP217 −5.14 23.4 ASP217ASP214ARG243

−4.42 19.0 ASP217 −5.83 10.1 ARG212 −5.19 9.2 ARG212

13. 3A4. Testosterone Dextromethorphan −4.49 18.8 ASP217 −4.71 17.8 ASP214 −4.42 19.0 ASP217 −8.03 12.5 ALA370 −5.19 9.2 ARG212

14. 3A4. Diazepam Dextromethorphan −4.42 19.0 ASP217 −4.36 14.2 PHE435 −4.42 19.0 ASP217 −6.97 12.2 ARG212 −5.19 9.2 ARG212

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Table 15 Drug-drug Interactions (Continued)

15 3A4. Piroxicam Midazolam −4.50 14.4 PRO429 NA −4.62 12.1 ARG372 −9.32 9.2 SER119 −7.09 5.2 ARG212

16. 3A4. Piroxicam Triazolam −5.76 11.7 GLY436 NA −6.30 11.4 GLU374 −8.55 9.1 ARG212 −7.75 11.0 ARG372

17. 3A4. Budesonide BROD −4.76 23.4 PHE220THR224

NA −5.45 6.7 ARG212 −6.64 7.9 PHE215GLY481ALA370

−7.44 4.0 SER312LEU483

18 3A4.Clotrimazole BROD −5.24 12.9 PHE215THR224

−4.80 19.2 THR224 −5.45 6.7 ARG212 −7.95 8.5 GLN484LEU483

−7.44 4.0 SER312LEU483

19. 3A4.Terfenadine BROD −4.61 17.4 VAL240 NA −5.45 6.7 ARG212 −5.92 12.8 PHE213 −7.44 4.0 SER312LEU483

20. 3A4.Diazepam BROD −5.27 12.6 THR224 NA −5.45 6.7 ARG212 −7.46 11.4 SER119 −7.44 4.0 SER312LEU483

X – Distance between Fe and reaction center (Å), E – lowest energy (kcal/mol)Grid 1: Firstly, the top-ranked conformer of modulator from blind docking is taken and then, the substrate is also blind dockedGrid 2: The nth rank of blind docking of modulator, which coincides with the substrate’s preferred (lone presentation) binding locus, is taken as the rigid docking template and then the substrate is blind docked next2C9.Flurbiprofen - the 3rd cluster (56th ranked) is similarly presented inside active site with energy of −5.33 (4.1 Å) as in 2° docking with DapsoneKey: Underlined S. No. indicates inhibition, punctuated S.No. indicates activation and bold S. No. indicates concentration dependent effectsaSubstrates which have the same binding site as their corresponding inhibitor/activator in their individual blind docking

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dependent effect, the active site based reaction mechan-ism does not provide any consistent explanation either.The most celebrated example of 9 shows clearly that thesubstrate’s reaction centre goes much farther than theinstance lacking the modulator. It is difficult to thinkthat activation could also arise because of multiple mole-cules’ simultaneous presence in the active site. Further, itis difficult to imagine how the presence of a modulatorcould affect minute changes in docking modalities,which could in turn enhance rates by ~400 % in 10 & 11(Kenworthy et al. 1999). In 7 & 8, contrasting results areseen for the same experimental effect of concentrationdependent activation/inhibition. That isin 7, the pres-ence of modulator makes binding energy more negativeand Fe-substrate reactive moiety distance decreases;whereas in 8, the presence of the very same modulatormakes the binding energy more positive and the Fe-substrate reactive moiety distance increases. In at leastfour cases (1, 9, 10 & 11), the substrate could still inter-act with the same key amino acid residues within the ac-tives (in spite of major or minor change in presentationmodes).Blind dockings of various substrates/modulators show

diverse binding loci on CYPs’ surface or regions adjacentto proximal/distal cavities. The distance of the reactioncentres are diverse and so are the binding energy terms.In certain instances, binding loci are conserved acrosssubstrates (Diclofenac and Flurbiprofen bind at Lys 72)and modulators (Dapsone and quinidine bind at Trp212), as exemplified by 1 & 9 for CYP2C9. This type ofparadigm was also seen for many molecules fromStatins, Sartans and NSAIDs classes for CYP2C9.(The respective images are shown in Additional file1A, Figure A1N 1.) In blind docking of individualmolecules (Table 15- as seen from 1, 6, 9 & 11- com-binations of different CYPs, substrates and modula-tors), the substrate or modulator molecules bind tothe same respective loci within CYP3A4. (Additionalfile 1A, Figure A1N 2) Even in blind docking with re-spect to Grid 1, while instances like 6 & 9 (inhibitionand activation respectively) could be explained by “ac-tive site” considerations, cases like 1 & 11 (inhibitionand activation respectively) speak against the erstwhileassumptions. This is further consolidated by the Grid1 data in cases 2, 3, 4, 5, (for inhibitions) 7 & 8 (forconcentration dependent inhibitions/activations) and10 (for activations), where the substrate binding doesnot get perturbed significantly by the presence ofmodulator. Even more, Grid 2 blind dockings, it canbe seen that in 2, 3 & 4 (for inhibition), 10 (foractivation) and 7 & 8 (for concentration dependentactivation/inhibition), the outcome cannot be ex-plained by a heme-pocket binding-based phenomenaalone. (The binding energy changes remain similar

and the Fe-substrate reaction centre distances areunfavorable.)Budesonide and testosterone have very similar struc-

ture but show varying effects on dextromethorphanmetabolism. Budesonide acts as an inhibitor whereastestosterone acts as an activator. From the entries in 12and 13 of Table 15, it can be seen that the presence ofboth modulators had very similar effects on dextrome-thorphan binding across all grids, thus failing to explainthe activation and inhibition seen. In another case, thesame modulator has varying effects on substrates havingsimilar structures (15 and16). Piroxicam inhibits Mid-azolam whereas it activates Triazolam metabolism. Thisis when the docking shows that presence of Piroxicamshifts both of the substrates to the same locus (withsimilar binding energy). Once again, the erstwhile para-digm does not explain the differences in the activityseen.In the cases of modulators studied 17 through 20, it is

known that Clotrimazole fully inhibits BROD metabol-ism whereas Budesonide, Terfenadine and Diazepam ac-tivate the same by several folds (Kenworthy et al. 1999).In active site docking, the presence of activators pushesthe reaction site in the substrate from 4 Å to ~8–13 Årespectively (in some cases, even farther than what the“fully inhibiting Clotrimazole” does), making it non-viable for a more effective direct oxygen rebound. Theactivation of Dextromethorphan metabolism broughtabout by Diazepam (14) also follows the paradigm seenabove.Most modulators studied had either a heterocyclic or

free amine nitrogen atom, possessing a lone pair.Thereby, they could be potentially capable of affectingthe heme centre by a direct Type II interaction. Table 16shows that all modulators’ (or substrates’) forced bindingat the distal heme pocket of the concerned CYPs gaveFe-interactive (reactive) moiety distance ranging from 4to 20 Å, non-conducive for a direct ligation or oxygenrebound. The distal pocket site docking results shownfor the modulators (Table 16) indicate that none of themolecules (except perhaps 9, 10 and 16) present them-selves in a favorable way, towards this outcome. Whilethe active sites of the concerned CYPs offer space to ac-commodate a majority of the modulators, some largemolecules (like 2) are docked with a significant partinteracting freely with bulk solvent. Further, the orienta-tions of the molecules were many times inappropriatefor a preferred modality of substrate activation/inter-action. The entries 13 through 16 are not discussed inTable 15 but they are important with respect to Houstongroup’s data with CYP3A4 (Kenworthy et al. 1999).[Gentamycin did not significantly inhibit or activate anyof the 10 substrates of CYP3A4 (or CYP1A2 catalyzedmetabolism of EROD). Nimodipine and Nitrendipine


(like Nifedipine) inhibited all substrates (loweringCYP1A2 activity for EROD marginally). Roxithromycininhibited some and did not perturb others (leavingCYP1A2 activity unperturbed).] The binding of thesemolecules at the distal site of CYP3A4 did not affordany insight upon the experimental effects observed. An-other important aspect to note was that azoles likeClotrimazole, Itraconazole and Ketoconazole loweredCYP3A4 activities for all substrates, when all of these‘azole’ modulators did not possess effective coordinationabilities (via the heterocyclic nitrogen) with the heme-

iron centre (as seen in entries 1 through 3 of Table 16).Only Clotrimazole inhibited EROD activity of 1A2 andwe explored blind docking to see if the ‘azoles’ bounddifferently with the two CYPs. Blind docking of Clotri-mazole to CYP3A4 was at a locus quite adjacent to theCYP’s proximal thiolate and only Itraconazole showedfavorable binding in the distal heme pocket (Additionalfile 1A, Figure A1O). In CYP1A2, the ‘azoles’ docked ata superficial crypt. It is highly unlikely that Clotrimazoleor the other large azoles (Ketoconazole & Itraconazole)gains access to the heme distal pocket at low

Table 16 Binding of modulators with CYPs

S.No.

Ligand Enzyme Dimension/Vol. (Å/Å3)



Distance(Å)


Distance(Å)


1 Clotrimazole 3A4 5.91, 5.66;311

−3.83 10.1 GLY436a −5.30 10.2 ARG212a -

1A2 −3.14 36.3 LEU51a nd nd nd

2 Itraconazole 3A4 11.32, 6.73;529

−5.31 27.6 PRO227 −4.18 21.1 Val240 LYS173TYR307 SER311

-

1A2 −3.50 33.7 ILE241 nd nd nd

3 Ketoconazole 3A4 11.55, 5.40;453

−3.78 29.1 VAL240a −6.64 10.5 ARG212 -

1A2 −3.69 37.5 VAL54 nd nd nd

4 Hydroquinidine 3A4 6.72, 5.86;320

−5.35 4.8 ARG212 −6.70 9.8 ARG212 (+)

5 Quinidine 3A4 6.26, 5.70;314

−4.19 11.2 ARG105a

ARG212a−6.56 9.2 ALA305 ARG212 -

6 2C9 −5.33 18.4 TRP212 −7.01 9.0 LEU208 -

7 Fluvastatin 2C9 6.94, 6.24;383

−5.05 19.6 PRO221 LYS72 −6.80 7.4 ARG108 ASN204 (+)

8 Fluconazole 2C9 5.35, 5.72;247

−3.88 4.0 ARG108a −4.65 7.6 GLY296 ARG108 -

9 Dapsone 2C9 6.30, 4.41;211

−4.36 20.9 TRP212 −5.11 3.2 THR301a

ALA297a+

10 Diazepam 3A4 6.10, 5.29;246

−5.64 9.3 ARG105 GLU374 −6.50 5.0 ARG212 +

11 Terfenadine 3A4 8.61, 5.79;487

−4.98 17.7 ARG372 −8.33 10.6 ARG212 -

12 Budesonide 3A4 7.77¸ 5.98;406

−5.10 7.7 ARG212 SER119ALA370

−6.89 7.7 ARG212 SER119ARG372 GLU374

(+)

13 Gentamicin 3A4 8.36, 5.99;476

−1.86 16.8 VAL240 CYS239 −4.77 10.8 ARG212 ARG372 -

14 Roxithromycin 3A4 9.16, 7.03;829

−0.46 24.8 LYS209 ARG243ASP217

+2.67 6.9 PHE213 ARG212GLU374

(+)

15 Nimodipine 3A4 7.48, 5.80;376

−3.79 25.1 VAL240 CYS239ARG243

−8.45 10.0 ARG212 ARG375GLU374

-

16 Nitrendipine 3A4 6.90, 5.65;318

−4.89 13.7 ARG212 ARG106 −7.51 5.8 ARG212 ARG375GLU374

+

17 Piroxicam 3A4 8.02¸ 4.83;269

−4.78 10.0 ARG106 −6.71 12.2 ARG212 ARG372 -

Dimension /Volume - maximal projection radius (Å), minimal projection radius (Å); Van der Waals volume (Å3)- bad presentation, + good presentation, (+) moderate presentation (not optimal but not facing the opposite end either)aNo H-bonds or pi-stacking interactions (neighboring amino acids provided)


concentrations of the enzyme. While the in silico bind-ing of CYP3A4 shows that Ketoconazole binds effect-ively at the surface of CYP3A4, the crystal structureshowed two molecules docked within the heme distalpocket (Ekroos and Sjögren 2006). When compared tothe drug metabolism data, the crystal structures do notmake much sense because Ketoconazole’s primarymetabolizer is CYP3A4 (Fitch et al. 2009). Clotrimazole,a facile one-electron redox active molecule, could evenalter the redox status within the microenvironment bydirectly interacting with superoxide/radicals in freesolution.

Docking of methylstyrenes (for reactions involvingoxygen insertion across an activated benzylic doublebond) with hemeproteins and the issue ofenantioselectivityIt is known that the crystal structure allows the explan-ation of stereoselective reactions in chloroperoxidase, afungal soluble P450 (Sundaramoorthy et al. 1998). Dy-namic movements of certain residues have also been re-ported to be responsible for the enantiotopic recognitionnear the heme centre (Morozov et al. 2011). The con-formation of the heme distal pocket is known to critic-ally affect the enantioslectivity involved in CPO. Theresults of docking of cis-betamethylstyrene with CPO/P450cam (Table 17) were compared with para-methyl-styrene’s aziridination using mutants of P450BM3. InCPO, the presentation of the substrate is close to theheme-iron. Experimentally, this reaction gave high en-antiomeric excess (ee) with high product yield (and thiswas in spite of the relatively harsh reaction system withlow pH and high peroxide concentration). In P450cam(with a relatively mild reaction condition at neutral pHand reductant), the access to heme centre is not ascloser (but binding energy was marginally better) and asa result, relatively lower ee was observed. (There was noinformation provided on the yield of the product orside-products formed in the reaction. Since P450camgives side-products even with camphor and its analogs,cis-betamethylstyrene should be no different.) This maybe an indication that the oxygen transfer is not strictly

mediated at the heme centre in P450cam. The recentpublication from Arnold group (Farwell et al. 2015)reported the bulky tosyl azide moiety insertion acrossthe styrene’s double bond in an aziridination reaction.This reaction is similar to the oxygen insertion acrossstyrene’s double bond (which gives epoxide). It is seenthat the efficient mutant (that affords higher ee andproduct yield) allows proximity of the double bond tothe heme centre and also gave better yields. Therefore,docking with the plastic crystal structure does give somequalitative idea about interactions of substrate at theheme floor in CPO, P450cam and P411BM3 (modifiedP450BM3).

Can detachment of products from the distal heme pocket(‘active site’) serve as the end of catalytic cycle (as soughtby the existent hypothesis)?Table 18 compares the centred and blind docking datafor some CYPs with their substrates and primary prod-ucts. The binding energy terms of the substrates arecomparable to the products, and at times (as is seen incase 1), the products have better binding energy termsthan the original substrate. The interaction of substrateand product is very similar for a given enzyme-substratecombination. Some products of a given CYP have morefavorable binding energy than another substrate of thesame CYP. These simple findings question the assump-tion that hydroxylations prompt the substrate to leavethe active site, owing to a lowering of affinity. Quite sim-ply, there is little chemical logic for the “committed sub-strate” to leave.

Meta-analysis of kinetic and equilibrium constants,residence and reaction timesWe analyzed the kinetics data retrieved from literaturefor two prominent liver microsomal CYPs. As shown inthe Table 19, the KM (and Ki) for diverse substrate(s) ofCYPs 2C9 and 2E1 (determined by different workers, invarious reaction systems) ranges between a few micro-molar to several hundred or thousand micromolarranges, which is an “unacceptable” spread in the value ofconstants.

Table 17 Heme-distal pocket centred docking of methyl styrenes with some non-microsomal hemeproteins (CYPs)

No. Substratea Enzyme LowestEnergy

Distance Interactions Presentation EE ofprdt.

Yield Rate Ref.

1 CBMS CPO −4.69 3.5 GLU183b PHE103b + 96 % 92 % na (~10/ min) (Allain et al. 1993)

2 CBMS P450cam −5.60 6.9 TYR96b THR101b - 78 % na ~1/min (Ortiz de Montellanoet al. 1991)

3 PMS P411BM3-CIS-T438S$ nd nd nd 25 % 1.1 % na (Farwell et al. 2015)

4 PMS P411BM3-CIS-T438S (I263F) −4.42 3.0 ALA264 55 % 40 % na (Farwell et al. 2015)aCBMS cis-betamethylstyrene, PMS para-methylstyrene, Energy and Distance given in kcal/mol & Angstroms respectively. bNo hydrogen bonds or pi-stacking butneighboring amino acids provided. Docking of 4 with tosyl azide gave docked molecules within 2.7 A of the heme iron, with ~ −4.15 kcal/mol energy term and itinteracted preferably with ALA330. Docking was not done with 3 owing to unavailability of protein structure


Further, we analyzed the value of Kd (as determined byin silico binding and in vitro protein-solution binding)and compared it with experimental KM (Table 20). Wesee little correlation between experimental Kd and ex-perimental KM. (At times, a theoretical breach occurswhen some experimental Kd values are larger than ex-perimental KM values, as seen in 3, 9, 10 & 12. Also, it isdifficult to imagine a several folds higher experimentalKM in comparison to Kd, as is seen in 2, 5, 8, 11.) Exceptfor 9 & 12, there is little correlation between experimen-tal Kd or experimental KM with in silico Kd (distal pocketdocked). But, even in 9 & 12, we have a theoretical fal-lacy that experimental KM is smaller than experimentalKd. This cannot be disregarded merely as an inefficiencyof docking programs. In the blind dockings, the in silicoKd values are higher than the experimental Kd. (Pre-dicted solubility or log P values are not highly differentfrom experimental findings.) There exists no correlationbetween residence time (as calculated from in silico Kd

or experimental Kd or experimental KM) with experi-mental conversion time. The magnitude of conversiontime is higher than the residence times by 103 (lowestlimits as seen in 8 and 9) to 108 (higher limits as seen in3 and 12). These differences are not trivial or small andtherefore, the “committed to catalysis” hypothesis mustbe rendered invalid. If we have to buy that proposal, weshould also accept that the most inefficient or poorsubstrates have the highest binding efficiency to a givenCYP! This cannot be supported with any in silico or ex-perimental data anyway. (Further, it counteracts thevery purpose for which the hypothesis was invented.)

The overall conversion time is low (<10 seconds) for“smaller and leaner” molecules with relatively unob-structed reaction site and it is not low for tight binders(lower Kd, whether in silico or experimental) or morereactive molecules/centres. This could show diffusionconstraints in reaction kinetics.

DiscussionEstablishing the need for this workIn the current work, we investigate the application of the“plastic crystal structure” (as available from pdb files de-posited by other researchers) to afford insight into thepurported “elastic dynamics” of CYP-substrate interactivemechanism. At the outset, we must state that exploringthe dynamic interactions of a squishy “breathing” proteinlocated in a semi-mobile phospholipid environment witha static protocol like molecular docking is not necessarilya very apt method. But then, one simple question contin-ued to bother us- if the F and G loops move to accommo-date all substrates of a given CYP within the heme distalsite, why is it that only some substrates are reacted by the‘high potential Compound I’ of a given CYP? Further, ourzeal to undertake the current study was fueled by the curi-osity generated upon noting the inadequacy of the erst-while hypothesis. It is known that reaction dynamics ofseveral CYPs do not show a classical Michaelis-Mentenparadigm for initial kinetics under steady-state conditions(Atkins 2005). Furthermore, several aspects (as notedfrom literature) speak out against a mere heme-centredactive-site hypothesis:

Table 18 Comparing the docking of substrates and products

Enzyme(PDB ID)

Ligand Blind docking Centred docking




3A4 (2J0D) Erythromycin −4.48 15.9 PHE304 −6.43 8.4 PHE304GLU374

(+)

Nor-Erythromycin

−5.37 6.7 PHE304 −7.91 11.8 PHE304GLU306

-

2C9 (1R9O) Flurbiprofen −5.86 17.8 PHE100LYS72

−6.59 4.6 ARG108ASN204

+

4’Hydroxyflurbiprofen

−5.52 15.8 PHE100LYS72

−6.17 4.1 ARG108ASN204

+

2C9 (1R9O) Diclofenac −6.15 18.8 LYS72 −6.39 17.0 ARG108 -

4’Hydroxydiclofenac

−5.37 20.2 LYS72 −5.28 9.1 ARG108 +

2E1 (3E6I) Chlorzoxazone −4.12 27.6 LYS486LEU463

−5.49 8.2 ALA299 (+)

6’Hydroxychlorzoxazone

−3.94 28.4 LYS486LEU463ASP470LEU471

−4.75 6.4 ALA299THR303

-

- bad presentation, + good presentation, (+) moderate presentation (not optimal but not facing the opposite end either)


1. Quite interestingly, upon changing the reductant orby the introduction of Cyt. b5 / lipids (as per theerstwhile understanding, these components shouldhave little influential scope to govern the substrate-CYP interactions), the KM value changes signifi-cantly (Parashar et al. 2014). This was when KM wascalculated using non-linear regression with only thelower values of the substrates (that fit the Michaelis-Menten paradigm), giving a curve that apparentlyresembled a decent hyperbolic asymptote. Also, it iscommon knowledge to many CYP researchers that

the overall stoichiometric yield varies with time andalso by altering the components’ initial concentra-tions. Such observations cannot be explained if thekinetics is critically dependent on a tight enzyme-substrate complex formation (as the erstwhilehypothesis solicits).

2. Kinetic Isotope Effects (or KIEs) afford us keyinsight into the dynamics of substrate and enzyme(reactive) intermediate’s interactions. The presenceof high intra-molecular KIEs (Miwa et al. 1980) inCYP-catalyzed reactions imply that there is little

Table 19 Kinetic constants for CYPs, as noted from literature

CYP2C9Substrate

KM Ki Ref. CYP2E1Substrate

KM Ki Ref.

Diclofenac 5.1 ± 0.9 μM(Baculosomes)

(Kumar et al. 2006) 4-Nitrophenol 120–140 μM (Hanioka et al. 2003)

2.6 ± 0.3 μM(Supersomes)

1.6 μM 21 μM 42 ± 19 μM (Tassaneeyakul et al.1993)

16 ± 2 μM,30 ± 3 μM(Reconstituted)

9.3 μM 108 ± 18 μM (Baranová et al. 2005)

4.0 μM (HLM) 2.9 μM (Walsky and Obach 2004;Kumar et al. 2006)

28 μM (Chen et al. 1996)

71 μM (Ngui et al. 2000) 197 μM (Duescher and Elfarra1993)

8.3 (Masimirembwaet al. 1999)

1.84 ± 0.09 mM (Fairhead et al. 2005)

9 μM (Bort et al. 1999) 156 μM (Van Vleet et al. 2001)

3.44 ± 0.45 μM (Konečný et al. 2007) 86 μM (Patten and Koch 1995)

(S)-Flurbiprofen 16.2 ± 0.9 μM(Baculosomes)

(Kumar et al. 2006) Chlorzoxazone 250-300 μM (Hanioka et al. 2003)

21.6 ± 0.8 μM(Supersomes)

69 μM 47 ± 10 μM (Tassaneeyakul et al.1993)


193 ± 28 μM (Baranová et al. 2005)

1.9 μM (HLM) (Walsky andObach 2004)

33 μM (Chen et al. 1996)

~33 μM (Hutzler et al. 2003) 0.65 ± 0.08 mM (Fairhead et al. 2005)

1.9 ± 0.4 μM 4.7 μM (Tracy et al. 1995) 50 μM (Beckmann‐Knopp et al.2000)

~7 μM (Youdim andDodia 2010)

150 μM (Howard et al. 2001)

19.4 μM (Tracy 2006) 660 ± 18 μM (Shimada et al. 1999)

(S)-Warfarin 4.6 ± 0.3 μM(Baculosomes)

(Kumar et al. 2006) Nitrosodimethylamine 212 μM (Patten and Koch 1995)

13 ± 2 μM(Supersomes)

36 μM


66 μM (Patten et al. 1992)

3.7 μM (HLM) (Hemeryck et al. 1999) 59 μM

4.1 ± 0.6 μM (Rettie et al. 1992) 21.7 ± 1.5 μM

4.1 ± 0.9 μM (Lang and Böcker 1995) 22.3 ± 0.6 μM


Table 20 Comparison of in silico and in vitro reaction / binding parameters

S.No. CYP Substrate Physical features(in silico)

ΔG (in silico) Kd (in silico),(μM)

Res. time (insilico-calc) (μs)

Physicalfeatures (exp)

Kd (exp-ref) KM (exp-ref) Res. time(exp-calc) (μs)

Conv. time(exp-ref) (s)

Solubility, log p& log D

Distalpocket

Blind Distalpocket

Blind Distalpocket

Blind Solubility,log p

FromKd

FromKM

1 3A4 Testosterone 115 μM,3.37 & 3.3

−6.61 −6.10 14 33 72 31 81 μM, 3.32 36 (Farooq and Roberts2010)

100 (Yuan et al. 2002) 28 10 53 (Wang et al. 1997)

2 Erythromycin 625 μM,2.60 & 1.55

−6.43 −4.48 19 508 53 2 2725 μM,3.06

125 (Farooq andRoberts 2010)

78 (Wang et al. 1997) 8 13 545 (Wang et al. 1997)

3 Amitriptyline 16 μM,4.81 & 2.8

−4.75 ND 348 ND 3 ND 35 μM, 4.92 178 (McLure et al. 2000) 67 (Eugster et al.1993)

5.6 15 1091 (Venkatakrishnanet al. 2000)

4 2E1 Chlorzoxazone 17457 μM,1.94 & 1.9

−5.49 −4.12 92 935 11 1.1 5898 μM, 1.6 ND 40 (Yuan et al. 2002) ND 25 300 (Lee et al. 2006)

5 p-Nitrophenol 25879 μM,1.61 & 1.28

−4.86 −5.67 267 68 3.7 15 83387 μM,1.91

23 (Hartman et al.2013)

197 (Duescher andElfarra 1993)

44 5.1 375 (Duescher andElfarra 1993)

6 2D6 Bufuralol 136 μM, 2.99& 0.81

−6.13 −4.28 31 713 32 1.4 ND, 3.5 6.2 (Guengerich 2005) 10 (Yuan et al. 2002) 161 100 5.9 (Marcucci et al.2002)

7 2C19 Omeprazole 1039 μM,2.43 & 2.43

−6.51 −4.43 16 553 61 1.8 ND, 2.23 ND 3.7 (R) 8.2 (S) (Foti andWahlstrom 2008)

ND 270 (R)122 (S)

1.7 (R) 6.9 (S) (Foti andWahlstrom 2008)

8 2C9 Flurbiprofen 102 μM,3.94 & 1.38

−6.64 −5.73 13 61 76 16 33 μM, 4.16 0.13 (Hummel et al.2008)

29 (Hutzler et al. 2001) 7692 35 4.8 (Hutzler et al. 2001)

9 Diclofenac 15 μM,4.26 & 1.58

−6.39 −6.15 20 30 50 33 8 μM, 4.51 16 (Wester et al. 2003) 6 (Lewis et al. 1998) 63 167 0.7 (Liu et al. 2012)

10 Warfarin 153 μM,2.74 & 1.34

−6.34 −5.72 22 62 46 16 55 μM, 2.7 8.2 (Takahashi et al.1999)

5 (Yuan et al. 2002) 121 200 194 (Liu et al. 2012)

11 2A6 Coumarin 453 μM,1.78 & 1.82

−6.33 −5.83 22 52 45 19 876 μM, 2.07 0.3 (Yano et al. 2005) 2.1 (Lewis andDickins 2002)

3704 476 2000 (Li et al. 1997)

12 1A2 Ethoxyresorufin ND, 2.28& 2.42

−7.15 −5.96 5.5 42 181 24 ND, ND 5.3 (Lin et al. 2001) 1.7 (Lewis andDickins 2002)

189 588 2609 (Eugster et al.1993)

The in silico ΔG and Kd values are from our studies (except 3); Kd exp is from equilibrium dialysis or Soret differential spectral analysis- found in literatureKM is any selected value reported for the enzyme-substrate combination in literature and conversion time is the time taken for one molecule of enzyme to convert one molecule of substrate to the specific productThe numbers in the large brackets in the last few columns are the pertinent references

Venkatachalamet

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spatial constraints and the substrate is very free toapproach the enzyme-generated catalytic intermedi-ate. This finding means that the substrate is nottightly bound in a preferred orientation within theactive site and is free to tumble around. (This intri-guing result is particularly relevant for non-activatedhydrocarbons, which are the classical reactions ofhigh potential reactive intermediate of CYPs.) To ac-count for this contradictory finding, some apologistsof the erstwhile hypothesis have invoked upon a“committed to catalysis” idea (Lu 1998), which seeksthat while the CYP is bound to the substrate to ef-fect a change in redox potential, the substrate is yetfree to rotate within the “active site”. The proposalflouts Occam’s razor because then, each CYP needsto have some kind of sensor(s) and processing intel-lect to effect this proposition. So, the erstwhile hy-pothesis does seek a case of mutually exclusiveoptions (obligatorily bound and freely rotating sub-strate) at the same instant. [Let us remember thatexistence of intra- or intermolecular KIEs in CYPreactions are not an indication that the reactionsmust occur within the heme distal pocket either.]

3. At times, the experimental Ki and Kd values of aninhibitor (Type I or Type II) for CYPs have beenreported to be lower than the enzymeconcentrations taken for assay (Locuson et al. 2004;Locuson et al. 2003; Collom et al. 2008). To abiochemist, inhibition of a biological process affordsthe most fundamental mechanistic andphenomenological insight regarding the crucial stepsinvolved and therefore, this is a critical point thatspeaks against the erstwhile hypothesis.

4. Survey of substrate “preferences” of mostmammalian P450s (for the hydroxylation reaction)do not give any clear cut ideas of active sitetopologies of a particular isoform. When a CYP cankinetically differentiate between an R and Senantiomer of a substrate (Rettie et al. 1992), it ishighly unusual that most of the human livermicrosomal hydroxylations are not enantioselective(i.e., do not give products with high enantiomericexcess). Also, in exceptional cases whereenantioselective hydroxylations are observed, thereactions are not regioselective (Dayer et al. 1982).Hydroxylations of molecules at unactivated carbonatoms (in either aromatic or aliphatic substrates)seldom show any enantioselectivity. This is whenoxygen insertion reactions by the same P450s forsimilar or much smaller substrates but withactivated carbon or hetero atoms with relativelyhigher electron density at the reactive moiety(olefins giving epoxides, N- or S- atom containingsubstrates giving the oxides, substrates with a

sterically unhindered benzylic carbon givinghydroxylated products etc.) showed varying levels ofenantioselectivity, depending on the substrates.These observations do not quite go well with a“space filling - topography - reactive moiety”considerations for an active site process.

5. In most cases, the hydroxylation is always higher onthe energetically favorable atom on the substrate(making CYP metabolic reactions highly predictable,(Jones et al. 2002; Korzekwa et al. 1990), and not thesterically favorable one. (There are exceptions to thistrend. Yet, we deem this overwhelming pattern as a“clear tell” for the microsomal P450s’ reactionmechanism.)

6. Jones group’s work demonstrated that it is adiffusible radical species (t-butoxy) that bestapproximates CYP catalytic process in terms of KIEs(kinetic isotope effect) and LFEs (linear free energyrelationships) when compared to a heme-centredFe-pyr system (Manchester et al. 1997). This is a keymechanistic insight that cannot be ignored.

When considering the mechanistic scheme of CYPs,we deem spatio-temporal aspects to hold more criticalimportance. And when one solves a puzzle that livermicrosomal CYPs are, the larger pieces have to bepieced in first. We considered a possibility that some ofthe observations (open-closed conformers, substrates-bound crystals and Type II spectra etc.) could be coinci-dental aspects, which may be inconsequential in the big-ger picture. If we take the erstwhile “high affinitybinding at heme pocket hypothesis” to be binding, thenthe endeavor of determining crystal structures would berelegated to lesser significance because dynamic aspectsalone would determine reactivity. Under the purview ofthe erstwhile hypothesis, crystal structures of diverseCYPs do not show a discernible structure-function cor-relation. All CYPs’ structures must be “tinkered” bymodeling/simulation to explain their reactivities, with-out really explaining their substrate preferences. In thelight of the arguments above, considering our recentfindings (as cited in the last part of the introduction)and the results presented in this study, we could in-dulge in an idea that the trigger of a molecule bindingto the heme pocket need not be the sole sponsors thatafford selectivity/specificity in CYP mediated redox re-actions. With this supposition, we justify the evolution-ary mandate of CYPs and the inherent crystal structureof a protein also gets its due significance. We also thinkthat it is quite probable that a hydrophobic proteincould be crystallized out in a number of ways, undervarying conditions, in the presence or absence of someorganic molecules. Whether these structures have func-tional relevance is to be decided by experimental/


functional verification. Literature also shows that inmost cases, a given microsomal CYP’s substrate is un-able to induce the production of the same CYP (somesubstrates of CYP3A4 are notable exceptions). Thisshowed that there was no biological/evolutionary linkfor a given drug molecule’s topology to the expressionof the CYP that metabolized the same drug molecule.[Further, it is opportune to add a disclaimer that theexample of P450BM3, a reductase-P450 hybrid, whichgives very high rates and enantioselective hydroxyl-ations (Farwell et al. 2015), may not be very appropriatefor the liver microsomal CYP systems. In these systems,the distal pocket’s and the access channel’s aminoacids’ role in substrate positioning/reactivity has beenwell-explored and documented (Ravichandran et al.1993; Li and Poulos 1997). Here, the reduction ofheme could be an intramolecular process, with facileelectron tunneling and proton relay. In microsomalCYPs, the electron transfer is currently believed to beinter-protein, and that, over apparent distances of >15Angstroms. This seems a rather unlikely proposal].Therefore, we had several reasons to believe that ourfindings on the phenomenology involved in peroxidasesystem were very relevant herein, within microsomalCYPs. Therefore, the prima-facie case for thinkingbeyond the currently available paradigm is established.

Critical dissection of the erstwhile hypothesisThe prevailing explanation for CYP catalysis (whichseeks the localizing of the substrate at the distal hemepocket) solicits that-

(i.)the protein be highly flexible (high level of inducedfit) and this flexibility can be exercised in severalmodalities (given the diverse topographies of itssubstrates), and

(ii.)each substrate should have a specific locus (ormultiple loci) to bind on the protein surface orcrypts therein. Further, this substrate-enzyme‘binding event’ should serve as a molecular triggerfor four obligatory outcomes -

(a)alteration of redox potential of the heme centre,(b)“commitment of substrate to catalysis”- that is, the

substrate should not dissociate through thesubsequent steps of the catalytic cycle and mustremain ‘bound and localized’ within the distalpocket,

(c)presentation of the substrate within suitable bondingdistance at the heme centre, for oxygen rebound &

(d)a proton shuttle must deliver protons at the hemedistal site for substrate hydroxylation.

This must be orchestrated in a precise sequence ofevents, as follows- 1. First, substrate should bind to thedistal site of CYP and it should be concomitantlyfollowed by CPR binding at the protein’s periphery.Then, a molecule of oxygen should diffuse into the distalpocket of the erstwhile complex, thereby giving yet an-other ternary or quaternary complex. All these fastidiousrequirements mock Occam’s razor and simple notions ofprobability. It is difficult to imagine how a single proteincould/should evolve the “molecular intelligence” for themetabolism of diverse xenobiotics through a highly com-plex process, sans chemical or biological logic. Fromdata given in this work (particularly, Tables 2, 3, and 4),one would challenge the very premise of the requisitessought by the erstwhile paradigm. This is because thecrystal structure shows that a CYP like 2E1 does nothave any channels leading into the distal pocket. OtherCYPs have some channels but most of them have toosmall dimensions (the substrate molecular dimension isat least twice the constrictions of the channels withinthe protein) afford any probability for a direct diffusionof the substrate molecule to “the locus of catalysis” (thedistal heme centre). Further, some substrates are toolarge to be even accommodated at the active site itself,even if it were to open up in a reasonable way.But apologists of the erstwhile hypothesis could argue

that the classical model of P450Cam also shows no cavity,and yet, it is very well known that camphor does show aType I binding in the distal heme pocket. Therefore, if thesoluble protein of P450cam is “flexible” enough to allowmovement of camphor into the distal pocket, the mem-brane proteins like microsomal CYPs can also be envis-aged to be flexible. Though membrane proteins are seenas ‘squishy’ proteins, they are also known to bind ligandswith high specificity, thereby indicating a high level of sur-face plasticity. Very importantly, the hydrophobicphospholipid microenvironment can be imagined to be aconstrained and low-energy microcosm, which is onlysemi-fluidic. The distal pocket may be large in many CYPsbut access to it is definitely constrained. To imagine thatsmall amounts of diverse substrates could repeatedly findthe probability to - (i) bind outside and get transported tothe inside or (ii) squeeze their way in or (iii) the enzymewould welcome the molecules by opening and closing upthrough some undefined logic - is seeking repeatedsequence of events of low probability (with respect to mo-lecular dynamics). Even if some of these large moleculesgathered access to the heme floor, we envisage that it isdisadvantageous for a direct oxygen transfer to any hin-dered locus within framework of a rigid substrate, withinthe constrained heme distal pocket. We speculated that itmay not be mere coincidence that microsomal CYPs’ sub-strate selectivity or docking orientations do not correlatewith reactivity or yield.


For the enzyme catalyzed reaction (where E is enzyme,S is substrate and P is the product)-

E þ S↔ ES −− EP → E þ P

let us assume that k1 and k−1 are the forward reactionrate constant to form ES complex and the backwardbreakdown rate constant of ES, respectively; and k2 isthe forward breakdown rate constant of EP complex.Then,

k1 ¼ d ES½ � = dtð Þ : 1= E½ � S½ �ð Þ

Theoretically, for nM levels of CYPs and μM levels ofsubstrate (the usual working ranges for in vitro studies),we can have only < nM ranges of [ES] formed in >milli-seconds (which is the generally accepted “breathingtime” of proteins, the time that would be required for F/G loops/helices to move around). So, the k1 for this re-action alone can be lower than 107 M−1 s−1 or be up tothe theoretical maximum of 109 M−1 s−1. Experimentally,nM levels of CYPs give product formation with pseudofirst order rates equalling 1 per second (for efficientlycoupled baculosome systems in vitro). For such systems,the overall reaction cycle’s second order rate constantachieves diffusion limitations for this single step alone.So, the diffusional constraints and probabilistic barri-cades involved in an ordered (sequential) reaction ofmultimolecular collisions/complexations of at least fivecomponents (nM levels of CYP & CPR and micromolarlevels of oxygen, substrate & NADPH) are unaccountedfor. Quite simply, under the purview of the erstwhile hy-pothesis, collision/complexation frequencies of bulkyhydrophobic proteins and drug molecules cannotachieve such super-concerted orchestration of events(that exceed diffusion limitations) at the phospholipidinterface. This ‘kinetic’ argument is the most compellingquantitative logic against the erstwhile hypothesis.

A new hypothesis, its stimulus and cruxRecently, we have argued that the efficient Type II bind-ing of a ligand at the heme distal pocket is seen only athigh concentrations of the enzyme (into micromolarranges) and at very high ligand pressures (Parashar et al.2014). The same logic would be held viable for Type Ibindings too. In physiological conditions, it is highly un-likely that such high concentrations of enzymes andxenobiotic substrates ever occur, for the “P450cam typelogic” to be of any functional significance. Further, inour lab studies, we couldn’t note any significant spectralchange (a hypsochromic shift of Soret spectra or theemergence of the high spin signature band at 650 nm)for some CYPs that we checked (CYP2C9-Diclofenacand CYP2E1-Chlorzoxazone; at micromolar level of theenzyme and a ligand pressure of 1:100). Also, it is diffi-cult to imagine that diverse molecules of varying topog-raphy and electrostatics could bind efficiently to the verysame loci in the distal pocket or near the channel to betransported to the distal site. A simple pictorial under-standing of the erstwhile paradigm is shown in Fig. 2.The erstwhile hypothesis requires that in the transitionstate, the heme-Fe-O species is within bonding distanceof both C and H atoms of the substrate. In the extensivedockings we carried out and from several crystal struc-tures of CYPs available till date, one cannot find any evi-dence for this requisite. On the other hand, if approachor binding of substrate to a protein surface or cryptserved as a molecular trigger [as seen in the well-knownand explicable example of some lipases where a flapopens up upon the presence of a hydrophobic substrate(Grochulski et al. 1993)], then the different loci for di-verse substrate binding should be mobile and engineeredto swing around and position the bound substrate atheme centre. Quite simply, this is asking for too much“intelligence” from a simple protein molecule. Weindulged even this proposition but could not find anyevidence to this effect (Tables 2 and 3). Further, the datafrom sections 8 through 10 of results presented above

Fig. 2 The erstwhile mechanism of binding of substrate at a distinct locus (distant from heme centre) and oxygen rebound at heme centre: Theerstwhile mechanism seeks (i) binding of the substrate to the heme distal pocket, (ii) the generation of a localized two-electron deficient reactiveintermediate at the heme centre, (iii) transposition/translocation of the substrate within bonding distance in the transition state and (iv) dissociation ofthe product and outward diffusion of the same owing to lower binding affinity (after the completion of reaction)


give very convincing arguments against the erstwhilehypothesis.We propose the following probabilistic phenomeno-

logical hypothesis, which not only explains most of theexperimental observations and in silico findings, but alsojustifies Occam’s razor’s criteria. (This is, albeit, at theaesthetic expense of tolerating a “radical” hypothesis.)The essential aspects of the ‘radical’ hypothesis, which isalready introduced in our earlier works, (Gideon et al.2012; Manoj et al. 2010a; Manoj et al. 2010b; Parashar etal. 2014) are as follows- CPR produces diffusible speciesthat serve as one-electron equivalents (like superoxide),which bind with CYP and get stabilized. These catalyticspecies (whether bound or free) thereafter convert thesubstrates. In this hypothesis, the heme centre can be re-duced or activated in three different ways-

(i.)One-electron equivalents generated by CPR can berelayed to the heme centre through diffusiblespecies, via the proximal thiolate (and a molecule ofoxygen could subsequently bind to the distal hemecentre). A survey of the prominent microsomalCYPs shows that all these enzymes have a readilysolvent accessible proximal site (Additional file 1A,Figure A1P), which makes this process very facile.

(ii.)The presence of a suitable substrate could reducethe heme centre on its own merit (and a moleculeof oxygen could subsequently bind to the distalheme centre).

(iii.)Superoxide or hydroxyl radicals generated in situcould bind to the distal heme centre by diffusionthrough the distal channel.

The outcomes of all the three steps above would bethe same. Thus, if we indulged a hypothesis that thehydrophobic distal pocket of CYP hemes could serve as“a residence/stabilization zone” for reactive species (likehydroxyl or superoxide radicals), then the functional life-time of the same increases significantly. This suppositionis justified by a pioneering paper published in the late1970s (Blumenthal and Kassner 1979). While probingthe binding of azide to a model hemoprotein, they hadconcluded that enhanced polarity of the heme distalpocket confers oxygen binding (and auto-oxidation ofheme) whereas the anion binding and its stabilization isfavored in the hydrophobic heme pockets. Therefore, ifthe CYP is not present in the vicinity to stabilize the dif-fusible reactive species, the one-electron equivalents dif-fuse out into the aqueous phase and are lost to waterformation. If CYPs are present and if the substrate isbound on the surface (or even the heme distal pocket orany other crypts therein) of the CYP and available nearby,the probability of reaction goes up very highly. The rateenhancement is neither through a one site “lock & key”

mechanism (alone) nor through an “induced fit”. The en-zyme enhances rates by stabilizing the diffusible reactiveintermediate and by enhancing the probability of thereactive intermediate to find the substrate in/around itsvicinity. Therefore, there exists a definite “uncertainty”with respect to the exact locus of interaction of the sub-strate and reactive intermediate. To the best of our know-ledge, such a way of enzyme functioning is not yetadvocated. Therefore, we coin our proposal as ‘murburn’hypothesis (“mured burning”- connoting a mild and unre-strained burning in a confined microenvironment) andsuch macromolecules as murzymes (mediating unre-strained redox catalysis). The representation in Fig. 3 cap-tures the essential scheme for interaction of variousxenobiotic substrates with CYPs. In comparison with theerstwhile hypothesis (shown in Fig. 2), the new hypothesisdoes not seek a high affinity binding or translocation intothe deep-seated heme pocket for a direct heme Fe-O todrug molecule bond formation step. Nanomolar con-centrations of diffusible radicals could be envisaged tohave effective diffusion radius of several Angstroms(even up to nano-scales), given the hydrophobic pocketof CYPs. The reaction mediated in such a scenario canbe nonspecific or regiospecific and even be enantiose-lective. The case must be noted that most CYPshydroxylate diverse drugs at the most energetically

Fig. 3 Interactions of various xenobiotics and DROS with microsomalCYPs: There are distinct binding sites for the two substrates (DROS andxenobiotics) and these loci are not brought together by an induced fit,for a direct bond-formation (between the two substrates) in thetransition state. There exists an uncertainty regarding the exactlocus where the two substrates finally interact/react, as a probabilistic fategoverns the outcome. The xenobiotic substrate 4 could go through theoxidation at/near the heme centre and such reactions would be favoredat high enzyme:ligand concentrations/ratios. Other substrates wouldhave better probability of being reacted by the given CYP away fromthe heme centre. This may be owing to better binding per se (as insubstrate 1) or/and because of a more probable presentation to thereactive species as it emerges out from the distal heme pocket (as insubstrate 3). The reactivity of a molecule like two would be dependenton several factors


favorable loci within the substrate molecule (and notthe most spatially open centre). This specificity can alsobe achieved in suitable chemical controls in enzymereactions that involve diffusible species ((Manoj 2006)and our unpublished work). Also, if the hydroxyl moi-ety incorporation was completed in the heme distalpocket at the heme centre, then we cannot explain howa bevy of substrates get activated by additives (Hutzleret al. 2001; Kenworthy et al. 1999) or why somesubstrates are not metabolized by some CYPs. Multiplemolecules binding at the heme centre in a dynamicstate would be a low probability event (though one getscrystal structures of these types of complexes fromhighly concentrated systems). We have already ex-plained these findings for heme-peroxidases (Andrew etal. 2011; Gade et al. 2012; Parashar and Manoj 2012)and the same explanations could hold well here too.

Application of ‘murburn’ hypothesis to explain kineticsand mechanismIf we continue with the discussion on the Michaelis-Menten supposition (from the earlier section above)-

KM ¼ k−1 þ k2ð Þ = k1ð Þ ð1ÞAs per laws of equilibrium-

Kd ¼ k−1ð Þ= k1ð Þ ð2ÞIt is clear that-

KM ¼ Kd þ k2=k1ð Þ ð3ÞTherefore,

KMe Kd; when k−1 >> k2 ð4Þand/or

KMe Kd; when k2=k1ð Þ ¼ 0 ð5ÞNow, the erstwhile hypothesis assumes that once the

substrate is hydroxylated, it loses efficiency for theenzyme. Therefore, the forward breakdown of EP maybe comparable or efficient with respect to the backwardbreakdown of ES. In this case, the contribution of k2cannot be neglected. Therefore, we might have a highervalue of KM than that of Kd. But under any circum-stances, the value of KM cannot be lower than that ofKd. If one were to accept the ideas advocated byGuengerich group, most microsomal P450-substrateforward binding is very fast and only diffusion limited(k1 ~ 109 M−1 s−1). If that were true, it is highly unlikelythat the physical value of k2 can approach k1 and there-fore, supposition (e) tends to be the applicable scenario(as is true for many enzymes in normal environments).Under such scenarios, the data in Table 20 cannot be

explained by the erstwhile hypothesis. The new hypothesisoffers the following reasons for the same-

1. Experimental KM: It is a reflection of multipleevents: (a) binding of substrate with enzyme, (b)production and binding of diffusible radicals withheme centre, (c) reaction of diffusible radicals withsubstrate & (d) competition of half-reacted substrate(intermediates) with other reactive species withinmilieu. If we imagine such a scenario, we can explainthe poor correlation of Kd and KM. With the newhypothesis, we can also account for the changes inKM values under various setups of the same CYPand substrate combination.

2. Conversion time: It is an index of the overallprocesses listed in 1 above. It is not an index of“committed to catalysis” or binding of substrate toheme pocket at all.

[Besides the above, there could be systemic errors inthe protocols employed in P450 research with- a. Experi-mental Kd determination by Soret differential spectrum:The Type I binding could be mistaken for heme reduc-tion, both of which are associated with a similar Soretchange. (Only the high spin marker at 650 nm is conclu-sive of Type I binding associated spin shifts for FeIIIheme thiolates.) Further, the binding of hundreds of mi-cromolar substrates to micromolar enzyme is not a goodmeasure of the reaction environment (wherein, nMlevels of CYPs have very little probability to bind to mi-cromolar levels of substrates). b. Experimental Kd deter-mination by equilibrium dialysis: Non specific bindingson to microsomal and other membranes would givehigher values. c. In silico Kd determination by hemepocket centered docking: Binding of a small organicmolecule with a hydrophobic pocket gives similar ΔGvalues for a bevy of substrates and non-substrates withCYPs. This binding cannot be taken as an index ofsubstrate’s affinity to the heme pocket. In many cases,actual access to the cavity may not be available on thekinetically relevant time scale.]Our study implies that binding and reaction of the

substrate at the heme-centre [as exemplified and soughtby oxygen rebound from Compound I, (Groves 1985)]may not be an obligatory requirement for many CYPs-substrates combinations. (However, the study does notrule out the possibility either. Particularly, at high con-centrations of enzymes (more than micromolar levels)and substrates (hundreds of micromolar levels), hemepocket binding of small molecules would be a relativelyhigh probability event (Parashar et al. 2014). We wouldalso like to add a disclaimer here that the hydrogen atomabstraction mechanism is not being challenged.) Onewonders how the constrained and hydrophobic distal


sites of CYPs afford an efficient proton shuttle in thephospholipid microenvironment. The pKa of the sub-strate’s alkyl/aromatic hydrogens are way higher to be ofany relevance (and that of the corresponding product’shydroxyl protons are several units higher than reactionpH). The physiological/reaction pH affords protons at ameagre concentration of only ~40 nM. An analysis ofthe active site and cavities/channels of microsomalCYPs shows clearly that this requirement sought by theerstwhile paradigm is stretching Occam’s razor criteria(Tables 2 and 3). Based on our works till date, the reac-tion scheme of the CYP + CPR system could be minim-ally represented now as-

2O2 þ NAD Pð ÞH → 2O2�−

þ NAD Pð Þþ þ Hþ Primarily; CPR’s roleð Þð6Þ

RX þ O2�− þ Hþ→ ROH

þ OX� Primarily; CYP0s = milieu’s rolesð Þð7Þ

O2�−= OX� þ O2

�−=OX�=RH = OH−= NAD Pð ÞH= H2O2= Enzymes → Diverse fatesð Þ

ð8ÞRX þ O2 þ NAD Pð ÞH → ROH

þ NAD Pð Þþ þ OX− Overallð Þð9Þ

The first three equations depict the essential initiationprocess and the multiple competitions at later stages.Now, we can understand why experimentally determinedKM values show wide variations across reaction setups.The interaction of reactive species with the substrate isnot captured or limited to its (substrate’s) binding ofCYP at a particular site, but is more about its presenta-tion in the relatively complex reaction scheme. Theoverall equation (9) shows the macroscopic thermo-dynamic drive at the phospholipid interface. That is- theuncharged hydrophobic molecules react to give ionicand hydrophilic species. This eventuality, in turn, servesas driving force for inundation and washing away bywater molecules. As seen from equation (9), the systemdoes not require extraneous protons. Till date, there ex-ists no evidence for the consumption of a solvent protonat the heme centre. There is only proof for the incorpor-ation of extraneous hydrogen atom into the substrateand this step need not occur at the heme centre inCYPs. CPR’s activity generates protons and radicals inthe microenvironment. CYPs stabilize radicals generatedand the event of oxygen and/or hydrogen atom insertioninto the substrate could occur even outside the hemedistal pocket. The rapid reactions of radical specieswould serve to pull the redox equilibrium to the right,

and this is achieved by substrate oxidation (more effi-cient) or depletion of ROS (less efficient). The process isessentially constitutive and the coinage of murburn hy-pothesis signifies the radical process involving oxygen inthe vicinity of heme (or other such redox) enzymes. Therelevance of such a process in liver cells (and within cel-lular membrane interfaces in general) and its mandate inevolution needs to be probed further. Most importantly,the hypothesis makes a lot of kinetic sense. At an in-stance, nM levels of CYP/CPR could stabilize suprana-nomolar levels of highly mobile ROS/radicals withinmilieu (and this process is not ordered). The reaction ofthese intermediates with micromolar levels of drug mol-ecules can explain the overall kinetics.The new hypothesis is not countered by the fact that

mutations of key amino acids in the heme distal pocketcould deleteriously or positively affect activity (Butler etal. 2013; Hamdane et al. 2008; de Montellano 2015).This effect need not be brought about by substrate-binding alone, but it can also be owing to altered ROSdynamics (Gideon et al. 2012). If we could think of “outof the active site” solutions to explain the phenomen-ology of CYP reaction chemistry, there is ample scopefor the murburn concept to explain most aspects of CYPreactivity. Very importantly, the new hypothesis justifiesseveral key mechanistic findings and proposals fromHollenberg’s and Newcomb’s groups (Lin et al. 2012;Zhang et al. 2011; Zhang et al. 2013; Sheng et al. 2009;Newcomb et al. 2003a; Newcomb et al. 2003b),regarding-

(i.)mechanism-based inactivations: It was observed thatloss of activity resulted owing to the covalentmodifications of CYPs’ surface amino acid residues,by substrate molecules like Bergamottin,Clopidogrel, ethynylphenanthrene etc. and

(ii.)presence of multiple electrophilic reactive species inthe CYP reaction milieu.

The new hypothesis could justify the KIEs and LFEsobtained in several P450 reactions. A substrate boundon a surface crypt or even freely tumbling around nearthe enzyme could be subjected to catalysis by a diffusiblereactive intermediate. The propensity and differentiatedreactivity of very low amounts of this agent’s interactionwith various moieties of a substrate could explain theextrinsic and intrinsic KIEs (Manchester et al. 1997) andprovides room for multiple reactant species (Newcombet al. 2003b; Coon 2003), which address concerns like-“the Fe-Oxo species does not have the required potentialto activate some substrates”. Further, the new conceptaffords a greater scope for explaining the activation/in-hibition of substrate catalysis by diverse additives. Thenew hypothesis can also explain how constrained loci of


the substrate molecules could get hydroxylated whenmore open carbon atoms are left untouched by thehighly reactive intermediate. Depiction of the overalltopographies and distal surfaces of the major CYPs areshown in Figure A1Q of Additional file 1A. CYP3A4,with three channels and a large heme distal pocket is themost proficient of all CYPs (followed by CYP2D6 andCYP2C9) (as seen in Fig. 4). Coupled with its highlyhydrophobic surface (to which diverse xenobiotic sub-strates can transiently bind) adjacent to the channelopenings, the probability of the reactive intermediatemeeting a substrate molecule in the vicinity of the distalpocket goes up significantly. (The large volume ofCYP3A4’s distal pocket is not enough to explain itsversatility because CYP2C9 also has a comparablehydrophobic voluminous pocket. CYP2D6 (with a rela-tively straight and wide channel) could allow smallmolecules’ enantioselective oxidations within the distalpocket, as is seen for benzylic hydroxylation of bufuralol.

CYP2E1, without a channel that leads to the distalpocket, is a special case. It can be seen that this systemhas excess accumulation of ROS species and obligatorilyrequires Cyt. b5. (This scenario is akin to P450cam need-ing putidaredoxin for effective activity. This may be be-cause the probability for the ROS to bind at the hemecentre via distal side goes down significantly.) Perhaps,the proximal side could also be involved in some reac-tion modalities. A specific antibody knocks out a givenCYP’s activity because the antibody would bind to thesurface region where the substrate would preferablybind. A strongly Type II binding substrate (which showssimilar in silico and X-ray structures; like the largeazoles) could potentially bind effectively to the hemecentre with F & G loop opening at high enzyme-inhibitor concentration. Otherwise, they would be effi-ciently metabolized by CYPs because the probability ofheme access of a large molecule would be lower whencompared to their metabolism outside (Andrew et al.

Fig. 4 Visualization of channels leading to the distal pocket in the major CYPs (from the distal surface). The first two rows have the distal viewwith the heme falling on the plane of the paper and the images have been rendered 20 % transparent, to show the position of the deep seatedheme (in deep blue). The highly continuous hydrophobic helices have been marked red. Further, an amino acid lining the access channel (if any)is also marked. The lower rows show the structures of the major CYPs. The amino acids marked with green are salient ones that mark the entryto the channel(s) leading to the distal pocket, as seen from different angles. In these images, the heme can be visualized through the channel, asyellow stick frames


2011; Manoj 2006; Manoj and Hager 2008). This is thereason why a molecule like Itraconazole is metabolizedby CYP3A4. The pseudohyperbolic asymptotes (hithertotaken as Michaelis-Menten curves) are an index of thereactive intermediate’s interaction with the substrate.Depending on the reaction microenvironment (partition-ing effects) and redox status therein, the curves can beaffected significantly. This is why lipids, CPR/Cyt. b5amounts etc. can alter the values of the pseudo KM ob-tained. The very low Ki values obtained at times are anindex of the radical formation and stabilization at lowconcentrations of the enzyme/additive. Larger moleculesneedn’t get into the distal pocket the through narrow ornon-existent channels. Earlier, it was difficult to answerthe question: Why is it that a given highly reactive inter-mediate of a promiscuous CYP (which could accommo-date a given or several small molecule(s) in its activesite) not observed to experimentally catalyze the givensmall molecule substrate? Now, it is understood thatbinding at an alternate and more topologically preferredlocation would be needed to enhance the probability ofthe substrate meeting the reactive diffusible intermedi-ate. Protons are not needed at the heme centre with thenew hypothesis. Hydroxylations caused by a diffusibleradical species tend to be non-enantioselective and non-regiospecific. Both scenarios can change if the radicalcatches a bound substrate in a particular enantiotopicpresentation, while it emerges from the heme centre orif the substrate is small enough and could gather signifi-cant access into the distal channel. The kinetic prefer-ence of a particular substrate enantiomer (withoutaffording enantioselectivity of the product) is perfectlyagreeable with the new hypothesis. Reactivity of the mol-ecules themselves and presentations within or outsidethe heme distal pockets could be reasons for differencesin activity within a given class of drug molecules. Mostimportantly, drug-drug interactions and genetic predis-position to drugs can be better explained by the new hy-pothesis. Also, since the new perception does not solicita tight binding, products could be further oxidized with-out the necessity of being bound within the heme distalpocket.

Projections to ratify the new hypothesis1. A simple way to positively test the murburn hypoth-esis is to look for reactivity of several CYPs towards anon-substrate with the introduction of a substrate (ofsuitable redox potential) and vice-versa (and noting en-hancement/inhibition if any). That is- a non-substratecould get converted if a suitable substrate is added intomilieu. The activity towards a substrate could go up orget lowered if a non-substrate is introduced into milieu.The newly proposed hypothesis predicts a two step, one-electron route (whereas the erstwhile hypothesis is

supposed to proceed via a single step, two-electronroute) and workers should be able to ratify our predic-tion upon suitable selection of the CYP/substrate/modu-lator and tailoring of reaction environment. 2. Anotherway to test for the new hypothesis (or negate the erst-while hypothesis) is- Small drug molecules that do notserve as “naturally” functional substrates could be pre-sented to CYPs (for example- Chlorzoxazone to CYP2C9and Flurbiprofen to CYP2E1) and the proteins allowedto crystallize. If we obtain crystals of the protein withthe “non-substrate” drug molecule within the hydropho-bic pocket, it must mean that this CYP structure (andthe processes that led the substrate docking at thehydrophobic distal pocket) has little functional signifi-cance. 3. Enantiomeric excess, yield/specificity and prod-uct formation rates could be studied for a variety ofmolecules (with both activated and non-activated carbonas reaction centres; keeping the functionality of the react-ive moiety constant) by- (a) increasing size of the moleculeby substitutions away from the reaction centre (to probethe general diffusion constraints) & (b) introducing substi-tutions adjacent to the reactive centre (to see if reactivityoccurs at the heme floor)- to give insights on the uncer-tainties involved. 4. The murburn hypothesis seeks obliga-tory role of diffusible radicals and ROS in the reactionscheme. Therefore, we should observe- (i) a temporalchange in reaction stoichiometry/coupling (dynamics ofROS and competitions thereof) by varying the reactioncomponents’ compositions/concentrations. (ii) Suitableone-electron scavenging small molecules, ions or enzymesshould possess the ability to modulate/inhibit CYP/CPRreactions. (iii) It should be possible to experimentallysimulate CYP reactions without CPR complexations (withROS species alone or with non-specific redox partnersand substrates). (iv) It should be possible to approach thereaction paradigm in CYP chemistry with suitable chem-ical controls. Also, chemical (non-enzymatic) controlsshould give enhanced specificity/rates upon reaction sys-tem modification (along the lines shown in Manoj 2006)for the hydroxylation reaction. 5. Modulations of reactionoutcome/efficiency may be noted to various levels bychanging the concentrations of CYPs/CPR and othersmall molecule redox-active additives (by virtue of affect-ing the competing reactions). 6. Most importantly- theerstwhile theory solicits that substrate binding to CYP isobligatorily required for the latter to receive electronsfrom CPR, leading to activation of molecular oxygen atheme centre. This supposition could be tested with simplereaction controls lacking the substrate (in diverse CYP-substrate setups) and checking for ROS in milieu.

ConclusionsWe have already demonstrated earlier that catalysismediated by enzyme sponsored diffusible species can be


very specific (regarding the preference of substrates, lo-cation of action within the substrate etc.) and projectedthe importance of the same (Manoj 2006). In those reac-tions also, the reactivity was afforded at the most ener-getically favored locus, quite akin to CYPs. So, it is seenthat the CPO model is quite relevant to CYPs, in termsof some aspects of the overall phenomenology. Now,ROS in CYP reactions need not be seen as a result of aninefficient enzyme or inefficient substrate (or their com-bination, together with “badly chosen” or “unoptimized”reaction parameters) alone. Earlier, the binding of sub-strate molecules within the active site was supposed toaccount for and/or prevent such eventualities. This isnow seen to be a redundant concept. The liver micro-somes have no way of getting to know what substratesthey would meet. So, the evolutionary agenda is ad-dressed by the combination of CYP-CPR-NADPH-dira-dical oxygen (in lipid microenvironment). Herein, wehave provided meta-analyses, arguments and extensive‘in silico’ findings based on currently available dockingprograms (which use scoring functions for spatial com-plementarities, electrostatics/molecular force fields) tosupport the deductions that-(1)Well-defined spatio-temporal constraints are not

very evident in several CYP reactions. This finding ishighly unlike a phenomenon occurring at occluded cen-tres such as the heme distal site. Therefore, the hemedistal pocket may be relatively passive in ‘interactions’with several xenobiotic substrates. (2) The newly pro-posed murburn hypothesis explains key experimentaland theoretical aspects of CYP reaction system.

Additional file

Additional file 1: Supplementary information. (PDF 6122 kb)

Competing interestsThe authors do not have any competing interests to declare.

Authors’ contributionsAV designed and carried out the experiments and reported data. AP carriedout preliminary docking studies/methods with controls, large substrates andSartans, introduced AV to molecular docking and compiled the table onbinding constants/reaction times. KMM identified and defined the problems,analyzed and interpreted data, proposed conceptual explanations and wrotethe paper. All authors read and approved the final manuscript.

AcknowledgementsKMM dedicates this work to the fond memories of (late) Lowell Hager(UIUC). Reviewers’ inputs enhanced the readability of this manuscript and wethank them for the same. This study was powered by Satyamjayatu: TheScience & Ethics Foundation.

Author details1Formerly at PSG Institute of Advanced Studies, Avinashi Road, Peelamedu,Coimbatore, Tamil Nadu 641004, India. 2Formerly at Hemoproteins Lab,School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu,India632014. 3Satyamjayatu: The Science & Ethics Foundation, Kulappully,Shoranur-2 (PO), Kerala 679122, India.

Received: 7 October 2015 Accepted: 5 February 2016

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